DNA-binding proteins of the zinc-finger class

ABSTRACT

The invention is directed to the design of DNA-binding proteins (DBP&#39;s) with capabilities of binding to any predetermined target double-stranded DNA sequence. Disclosed are the rules for design of the proteins; an algorithm for screening for the optimal DBP&#39;s; a computer system employing the rules and the algorithm; general formulae encompassing the proteins; and methods of use of the proteins.

This application claims the benefit of U.S. Provisional Application No. 60/075,466, filed Feb. 20, 1998.

BACKGROUND OF THE INVENTION

A superfamily of eukaryotic genes encoding potential nucleic-acid-binding proteins contains zinc-finger (ZF) domains of the Cys₂-His₂ (C₂H₂) class. Proteins that have these characteristic structural features play a key role in the regulation of gene expression[1-4]. Sequence comparisons, mutational analyses, and a recent crystallographic investigation have revealed that each finger domain, as a rule, interacts with the major groove of B-form DNA through contacts with some or all three base pairs within a DNA triplet. These base-specific interactions are mediated through amino acid (AA) side chains at specific positions in the a-helical region [5-10] of the protein domain.

Although the AA sequences of more than 1,300 ZF motifs have been identified, the exact DNA-binding sites are known only for a few proteins. The available information on DNA contact regions concerns mainly guanine-cytosine-rich strands [5-9] and fewer adenine-thymine-rich sites [11,12]. On the basis of experimental data, the first proposals for rules relating ZF sequences to preferred DNA-binding sites have been made [13,14]. However, no general rules for ZF protein-DNA recognition have been proposed. This is likely due to the fact that neither computer modeling [2,3,5] nor crystallographic analysis [7] have provided enough information on the overall structural variety in the ZF-DNA contact region.

Using physical atomic-molecular models to characterize the steric conditions in the specific contact positions for different ZF-DNA interactions, an objective of the work leading to the present invention was to determine a set of general rules for ZF-DNA recognition for the C₂H₂ class of ZF domains. Once this objective had been reached, the work of the invention plan was to develop an algorithm, and a computer system using the algorithm, to design effective zinc-finger DNA-binding polypeptides. The achievement of these goals represents a major advance of knowledge in the field, knowledge characterized by the disclosures of Rebar, et. al. and Beerli, et. al. [15,16]. These two disclosures are concerned with the selection, using the phage display system, of specific zinc fingers with new DNA-binding specificities. On the other hand, the present disclosure is concerned with the design of DNA-binding proteins for any given DNA sequence.

SUMMARY OF THE INVENTION

The invention is directed to the design and specification of DNA-binding proteins binding via C₂H₂ zinc-finger motifs (DBP's or, individually, a DBP). On the basis of the studies described herein, general rules for optimizing such binding have been determined, and a formula describing the class of DBP's having optimal DNA-binding properties has been constructed. Furthermore, a program has been developed, based on the rules, which affords the design of DBP's with such high binding affinity for any given DNA sequence. Lastly, rules have been determined for the design of DBP's which, while not having optimal binding, do have significant and useful DNA-binding properties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the alignment of ZF domains in various known DBP's. TFIIIA fingers 1-9 are shown as SEQ ID NOS: 207-215, respectively, in the Sequence Listing; Xenopus Xfin finger 31 is shown as SEQ ID NO: 216; the ADRI finger is shown as SEQ ID NO: 217; MKR2 fingers 1-9 are shown as SEQ ID NOS: 218-226, respectively; and Kruppel fingers 1-5 are shown as SEQ ID NOS: 227-231, respectively.

FIG. 2 is a schematic representation of the interaction between a target DNA triplet and a single ZF domain.

FIG. 3 is a schematic representation of the interaction between a target DNA string of 9 bases and a three-domain DBP.

FIG. 4 is a block flow diagram of the computer system by which the instant DBP design process is implemented.

FIG. 5 is a block flow diagram wherein the Computer Program block (2) of FIG. 4 is further broken down.

FIG. 6 is a block flow diagram wherein the Process Genome into Blocking Fragment Files block (2) of FIG. 5 is further broken down.

FIG. 7 is a block flow diagram wherein the Design DBP's for a Genome block (3) of FIG. 5 is further broken down.

FIG. 8 is a block flow diagram wherein block (22) of FIG. 7 is further broken down.

FIG. 9 is a block flow diagram wherein block (24) of FIG. 7 is further broken down.

FIG. 10 shows the distribution of binding strengths of acceptable 9-finger DBP's across the yeast genes analyzed.

FIG. 11 shows the values of the binding energies of the acceptable 9-finger DBP's found for the yeast genes analyzed.

FIG. 12 shows the distribution of DBP subsite (spurious) binding energies across the yeast genes analyzed.

FIG. 13 shows, in nonlogarithmic fashion, the distribution depicted in FIG. 12.

FIG. 14 shows the ratios of binding energy to subsite (spurious) binding energy, across the yeast genes analyzed, for the acceptable 9-finger DBP's.

FIG. 15 shows the values of the spurious binding energies for each of the 27-base-pair (bp) frames of the 300-bp promoter region of yeast gene YAR073.

FIG. 16 shows the ratios of binding energy to subsite (spurious) binding energy for each of the 27-base-pair (bp) frames of the 300-bp promoter region of yeast gene YAR073.

FIG. 17 shows the distribution of sizes of acceptable DBP's across the C. elegans genes analyzed.

FIG. 18 shows the ratios of binding energy to subsite (spurious) binding energy, across the C. elegans genes analyzed, for the acceptable DBP's.

DETAILED DESCRIPTION OF THE INVENTION

The general rules governing the binding of C₂H₂ ZF motifs to DNA were developed by using a combination of the database analysis of the homologies between 1,851 possible ZF domains and physical molecular modeling of the interaction of a DBP model with a DNA model containing all 64 possible base-pair triplets. The DBP model approximates the size and shape of a half-gallon jug of milk. The DNA model is approximately four feet long and one foot in diameter. The axis of the DNA model is horizontal and can be rotated to observe each of the 64 base-pair triplets. By moving the DBP model in and out with respect to the DNA model one can observe the amino acid and nucleic acid contacts.

Although the following description details the scientific precedents of this invention, the completeness of the rule set governing the DBP-DNA interaction could have only been obtained by the continual, derivative interplay of data base analysis and physical modeling during the invention period. Observations as to the conservation and variability of amino acids at various places in the ZF motif were embodied, first, by constructing a physical model of the ZF motif and, then, by physically modeling the interaction of a specific DBP with a designated DNA bp triplet. The physical modeling indicated patterns of amino acid and nucleic acid interaction which led to further analysis of the database. Iterations of this interplay between database analysis and physical modeling enabled conceptual refinement and expansion of the nature of contact patterns. As these patterns emerged, systematic variation of the amino acids in the ZF motif was undertaken for each of the 64 base-pair triplets. The physical modeling of the interaction between a DBP and DNA was efficient because alternative amino acids could be easily introduced into the ZF motif and the resulting protein physically modeled against the DNA. Hydrogen bonding, and water and hydrophobic contacts could then be modeled, clearly determined and counted very quickly. From this physical modeling a general set of rules was developed which incorporates criteria for the design of DBP's that specifically interact with DNA.

The utility of ZF sequence analysis and alignment is illustrated by FIG. 1. The TFIIIA protein is widely used as a model for ZF proteins both in terms of physical measurement and modification and theoretical data analysis. For each of the nine zinc-finger domains the TFIIIA amino acid sequence in this figure has been aligned so that the zinc-binding amino acids, the two cysteines (CYS) and the two histidines (HIS), are aligned in four columns. In order to achieve this alignment dashes must be inserted into the sequence at various places to provide for domains which have additional amino acids. The same type of alignment has been done for ZF protein MKR2 and the Kruppel proteins. The MKR2 sequence alignment is very compact; there is no need for any insertions, since all of its ZF domains are of the same size. Compared to TFIIIA, MKR2 acts as a much more uniform model for studying the interaction of the amino acids of the protein with the bases of the specific double-stranded DNA. To arrive at the present invention, MKR2 has been used exclusively as the sequence basis for deducing the general rules which govern DBP-DNA interactions.

The crystallographic analysis of a complex containing three ZFs from ZF protein Zif268 and a consensus DNA-binding site helped identify the localization of ZF-B-DNA recognition sub-sites [7]. Because the mutagenesis and sequence investigation results are in accordance with crystal structure data, it is reasonable to expect that the same contact regions also participate in the interaction of other ZF-DNA complexes [5,6,8-10]. Thus, it has been assumed that the following ZF components of a ZF protein play a key role in the anti-parallel DNA reading process: 1) the AA immediately preceding the a-helical region of the protein; 2) the third residue within the a-helical region, i.e., that immediately preceding constant leucine; and, 3) the sixth residue of this region, i.e., that immediately preceding invariant histidine.

These components are indicated below as Z₃, Z₂ and Z₁, respectively, in the generalized ZF sequence (a-helical and b-structural regions are underlined) given in Formula I:

wherein X is any amino acid; X₂₋₄ is a peptide 2 to 4 amino acids in length; X₃₋₅ is a peptide 3 to 5 amino acids in length; X₀₋₂ is a peptide 0 to 2 amino acids in length and C, D, E, F, G, H, K, L, P, R, S, T and Y designate specific amino acids according to the standard single-letter code. Pairs of letters separated by “/” indicate that the position can be filled by either of the two specific amino acids designated.

Keeping in mind the above formula, one can envision the formation of antiparallel, trinucleotide-peptide complexes with three (first, second and third) contact positions as follows:

5′—N₁—N₂—N₃—3′

COOH—Z₁—Z₂—Z₃—NH₂

The crystallographic investigation of the Zif268-DNA complex also gave indications of the way the contact groups interact. Pavletich and Pabo [7] concluded that Zif268 forms 11 critical hydrogen bonds (H-bonds) with the bases of the coding DNA strand in the major groove. Two arginine residues in the first contact positions (see the designations of positions above) make H-bonds with the N7 and O6 atoms of the guanine. Three arginine residues hydrogen bond in the same way with guanine in the third contact position. In addition, each arginine residue in this position forms lateral H-bond, salt bridge interactions with carboxylate groups of aspartic acid occurring as the second residue in the a-helix. The N6 atom of the histidine residue in the middle contact position of the second ZF of Zif268 donates an H-bond to the N7 or O6 atom of guanine. The role of arginine and histidine residues in the interaction with guanine in ZF polypeptide-DNA complexes is confirmed by experiments of directed mutagenesis [5,6,9,14]. The crystallographic investigations of DNA-binding domains of lambda and phage 434 repressors, complexed with corresponding operator sites, revealed that guanine can also be H-bonded by lysine, asparagine, glutamine and serine residues [17,18]. No doubt, the remaining polar AA's—threonine and tyrosine—are able to form analogous bonds with guanine.

In fingers 1 and 3 of the Zif268-DNA complex, the second (middle) critical position is occupied by a glutamic acid that does not contact the cytosine at the corresponding region in the DNA [7]. However, ZF protein-DNA binding assays have shown that in natural binding sites this interaction does occur with both glutamic acid and aspartic acid [5,6,9,14,19]. Desjarlais and Berg [14] proposed an H-bonding formula for the interaction between cytosine and aspartic acid. The authors emphasized that the preference for aspartic or glutamic acid in the interaction with cytosine depends on the presence of glutamine or arginine in the third contact position (Z₃), and serine or aspartic acid in the second position (Z₂). The mutagenesis experiments of Nardelli et al. [5] reveal that cytosine can interact with a glutamine residue. This may also be true for asparagine, which has similar polar groups. Cytosine should also be capable of making an H-bond with the hydroxyl oxygen atom in serine and threonine residues.

Thymine in the Zif268-DNA complex does not seem to participate in the recognition process. However, the crystal structure investigations of the lambda repressor, DNA-binding-domain DNA and engrailed homeodomain-DNA complexes, as well as ZF protein-DNA binding assays, demonstrate that thymine can make both hydrophobic contacts with non-polar residues (alanine, leucine, isoleucine, valine) and H-bonds with polar AA's (lysine, arginine, glutamine) [8,11,14,17,20].

The X-ray crystallographic studies of lambda and phage 434 repressor, DNA-binding domain complexes with corresponding operator sites revealed that an adenine base forms two H-bonds to glutamine: 1) the amide NH₂-group of the glutamine side chain donates an H-bond to the N7-atom of adenine and 2) the amide O-atom accepts an H-bond from the N6 atom [17,18]. Similar H-bonds have been found between adenine and asparagine residues in the two homeodomain complexes [20,21]. ZF protein-DNA binding assays also indicate, that in ZF contact positions, adenine makes strong interactions with both glutamine and asparagine [8,11,12,14]. Considering that glutamic and aspartic acid carboxylic groups have O-atoms capable of accepting H-bonds as do glutamine and asparagine amide O-atoms, one may suppose that adenine can form a single H-bond with both glutamic and aspartic acid. Indeed, Letovsky and Dynan [19] have shown in a directed mutagenesis investigation that transcription factor Sp1, containing a glutamic acid residue in the central contact position of the ZF, binds only 3-fold more weakly to the adenine-substituted variant (-GAG-) than to the wild consensus recognition site (-GCG-). In addition, Desjarlais and Berg [14] and Berg [8] think it probable that adenine can (like guanine in the Zif268-DNA complex) make one H-bond to a histidine residue. It is likely that not only histidine but also other polar amino acids (arginine, lysine, tyrosine, serine and threonine) are capable of forming an H-bond to atom N7 of adenine.

A database of potential ZF protein domains, containing 1,851 entries, has been assembled. This database was used computationally to observe the homologies between the ZF domains.

Several years ago Seeman et al. [22] concluded that a single H-bond is inadequate for uniquely identifying any particular base pair, as this leads to numerous degeneracies. They proposed that fidelity of recognition may be achieved using two H-bonds, as occurs in the major groove when asparagine or glutamine binds to adenine, and arginine binds to guanine.

On the basis of the above-given results, it was reasonable to test, using the models described herein, base recognition at the ZF contact positions of the following AA's:

1) guanine—R, H, K, Y, Q, N, S, T;

2) cytosine—E, D, Q, N, S, T;

3) thymine—I, L, V, A, R, H, K, Y, Q, N, S, T;

4) adenine—Q, N, E, D, H, R, K, Y, S, T

Plastic space-filling atomic-molecular and ionic models [23,24] have been used to build ZF-DNA complex imitations. These molecular models were chosen due to the extraordinary firmness of their connectors, their convenient scale (1 cm=1 Å=0.1 nm) and their improved theoretical parameters which were very suitable for the modeling of macromolecules. New modules of tetrahedral carbon atoms, with bond angles 100° and 105°, dihedral oxygen atoms (120°) and tetrahedral phosphorus atoms (102° and 118°), maintained the exact modeling of deoxyribose puckering and sugar-phosphate chain conformation in the B-DNA model. Peptide bonds in the DBP models were imitated by the fixing, to each other, of special modules of carbon atoms (bond angles 116°, 120.5° and 123.5°) and nitrogen atoms (122° and 119°). The zinc ion was represented in the model by a sphere (R=0.85 cm) fixed tetrahedrally to N and S atom modules of ZF histidine and cysteine residues. A long horizontal 34-base B-form DNA model with laterally-fixed DBP models was used for docking experiments.

In the first stage of the subject investigation, the models of Zif268 fingers 1, 2 and 3 were assembled, and the general spatial orientation of the ZF-B-DNA complex was observed. In the second stage, the steric fitness of all 64 nucleotide triplets to the different combinations of the above-mentioned AA's in the critical positions of the ZF-DNA complex was modeled.

A plastic molecular model of the Zif268 peptide-DNA complex was assembled on the basis of crystallographic data [7]. After the imitating of ZF-DNA backbone contacts and H-bonds between AA and bases in the major groove, it was confirmed that the overall arrangement of Zif268 is antiparallel to the DNA strand. The most steady ZF-DNA, nonspecific interaction seems to be the H-bond between a phosphodiester oxygen atom and the first invariant histidine residue fixed to the Zn²⁺ ion. A conserved arginine on the second b strand also contacts phosphodiester oxygen atoms on the primary DNA strand. However, fingers 2 and 3 of Zif268 contact equivalent phosphates with respect to the 3-bp sub-sites, whereas the finger-1 H-bond is shifted by one nucleotide. Another four ZF-DNA backbone contacts made by arginine and serine residues are even more irregular in relation to the ZF modular structure.

All 11 critical H-bonds found in the Zif268-DNA crystal complex have been observed in the plastic models. As expected, the threonine residue in the first contact position of the second finger was too far from thymine to make an H-bond. However, differing from the results of crystal structure analysis, the model investigation clearly indicated the possibility of hydrogen bonding between a glutamic acid residue and cytosine in the second contact position of fingers 1 and 3.

It is noteworthy that, of the six guanine-AA contacts in recognition positions observed in the Zif268-DNA crystal structure, five were made with arginine and only one with histidine. It is even more interesting that this histidine-guanine interaction was the only one in the central-specific position. Considering the smaller size of histidine in comparison with arginine, it may be supposed that the middle position has steric constraints prohibiting contact between guanine and the larger arginine residue, although, due to its capability of forming two H-bonds, the latter pairing should be energetically favored.

To investigate the spatial conditions in different recognition positions, a B-DNA model was built which contained, in the primary strand, 1) the triplet GGG, and 2) models of ZF α-helical protein fragments (including the AA immediately preceding the a-helix) with a) side groups of the first Zn-binding histidine and b) groups for critical AA triplets R₁R₂R₃ and R₁H₂R₃. The models of a-helical fragments were fixed to the B-DNA model by an imitation of an H-bond joining a phosphodiester oxygen atom with a histidine residue. Specific base-AA contacts were then tested in these complexes. It was elucidated that only the complex GGG-RiH₂R₃ contains the contact groups in positions corresponding to the distances of critical H-bonds found in the Zif268-DNA crystal structure. The complex GGG-R₁R₂R₃ is sterically unfavorable; molecular modeling reveals that, although in the outer contact positions guanine and arginine can be joined by two H-bonds, in the middle position such a pair cannot be included due to the limited space.

Observations derived from the physical models confirmed the supposition of steric constraints for some AA-base contacts in the central contact position. In the case of the complex G₁G₂G₃—R₁H₂R₃, the following approximate distances from guanine N7 and O6 atoms to the C_(a) atoms of corresponding AA's have been determined: G₁N7—R₁=7 Å, G₁O6—R₁=8 Å, G₂N7—H₂=5.5 Å, G₂O6 H₂=6.5 Å, G₃N7—R₃=8 Å and G₃O6—R₃=7 Å.

Using the models, the investigation of B-DNA and a-helix basic structure elucidated the molecular basis for steric constraints in the second ZF-DNA recognition position. Joining, by a straight line, the analogous atomic groups (for example, N7 atoms of guanine) of the first and third base in the DNA triplet in the major groove results in the corresponding group of the middle (second) base being distanced from this line by about 1.5 Å. Similarly, joining the C_(α)atoms of the AA's in the first and third contact positions of the ZF by such a line results in the C_(α)atom in the middle position also being at a distance of about 1.5 Å. Thus, the space allowed for a critical AA in the middle contact position is compressed from both sides approximately 1.5 Å.

Analysis of the above-given data on the ZF-DNA backbone contacts, as well as observations derived from the models, led to the conclusion that there are considerable differences in spatial conditions between first and third ZF-DNA recognition positions. In the first position the C_(α)atom of the AA is distanced about 6.5 Å from the phosphodiester oxygen atom where the ZF protein is fixed to the DNA backbone by the invariant histidine residue. Due to the steady fixing of this ZF α-helical part by histidine, the freedom of conformational rearrangements in the first contact position is limited: the C_(α)atom, with corresponding side chain, can be moved 2-3 Å “up and down” in the plane of the base where it is localized in the primary DNA strand or, alternatively, 1-2 Å perpendicularly to this plane.

On the other hand, the fixing of the N-terminal end of the ZF α-helical region to the DNA backbone seems to be rather loose and variable, therefore allowing relatively large rearrangements for the C_(α)atom and the corresponding AA in the third contact position. The latter contact position is favored by the fact that the C_(α)atom in this position is more distant from the main fixation place (about 10.5 Å from the phosphodiester atom bound to the histidine residue), and the corresponding AA in this position is not a part of the α-helix. The most important finding is that, due to the above-described circumstances, the critical AA in the third contact site can apparently occupy very different positions in the corresponding bp plane. This means this residue may, in certain complexes, be very close to the base of the complementary DNA strand. One of the reasons for the appearance of such a geometrical configuration is that the typical, right-handed helical twist of B-DNA makes the complementary base on the nucleic acid second strand in the third contact site even more accessible than the main base on the primary chain. Molecular modeling clearly shows that in the third, and also partially in the second contact position, this DNA strand is capable of participating in the ZF-nucleic acid recognition process. In the Zif268-DNA crystal complex, the α-helix of each ZF domain, which is bound only to the DNA primary strand, is tipped at about a 45° angle with respect to the plane of the base pairs [7]. In cases wherein the second DNA strand, via critical H-bonds involving the third and second contact positions, is involved in the reading process, the direction of the α-helix axis should be even more perpendicular to the base pair plane.

Thus, this more detailed investigation of ZF-DNA-complex imitations, through use of physical molecular models, shows that steric conditions in each of the three contact regions are different. These steric conditions are reflected in the ZF-DNA recognition rules.

On the basis of information obtained above, which yielded a general observation of steric conditions in the ZF-DNA recognition process, an extensive model study of various AA-base combinations in the critical contact positions was undertaken. The results of this investigation are presented both as the ZF-DNA reading code and main rules for recognition (Tables 1, 2 and 3). The rules are in good accordance with crystallographic, directed mutagenesis, DNA-binding and sequence analysis data.

With reference to the sequence of Formula I and the 2-dimensional structure diagram in FIG. 2 (which provides a schematic representation of a zinc-finger domain and its interaction with a DNA strand), the studies confirmed the identity of the three critical contact positions in a given zinc-finger domain as follows:

1) between the first nucleotide in the triplet and the first AA preceding the constant histidine at the COOH end of the α-helix;

2) between the second nucleotide in the triplet and the fourth AA preceding the constant histidine at the COOH end of the α-helix; and,

3) between the third nucleotide in the triplet and the seventh AA preceding the constant histidine at the COOH end of the α-helix.

Steric conditions in the three contact sites of the ZF-DNA recognition complexes are different. The first contact position is relatively large and strictly fixed, which enables the binding of a longer AA to bases on the primary DNA strand with sufficient specificity and affinity. The second position is compressed and can accommodate smaller AA's with somewhat lower specificity and affinity. The third position allows considerable conformational rearrangements including the contacts with the complementary DNA strand.

In Table 1, for each nucleotide of a given DNA triplet on the primary strand, both main (Column A) and alternative (Column B) base-binding AA's are presented. Both specificity and affinity were considered in including a residue in Column A. As was proposed already by Seeman et al. [22], the fidelity of recognition is better maintained, in the case of purine bases (guanine and adenine), because they occupy a greater portion of the major groove and offer more hydrogen bonding sites than the pyrimidines. Therefore, the strongest AA interactions appeared to be those of arginine, glutamine and asparagine, each binding by two H-bonds to either guanine or adenine. The affinities of aspartic acid, glutamic acid, asparagine and glutamine were frequently enhanced by the formation of water bridges between carboxylate or amide oxygen atoms and DNA backbone, phosphodiester oxygen atoms. Although van der Waals interactions are relatively weak, they can play a certain role in recognition of the thymine methyl group by hydrophobic AA's (alanine, valine, leucine and isoleucine).

As indicated in Table 1, in many ZF-DNA complexes the base recognition in the nucleotide triplet of the primary DNA strand occurs not entirely via the primary strand, but by binding simultaneously to both the primary and complementary strands, or even exclusively to the complementary strand. Without “help” from the complementary DNA strand, the binding of critical AA's to nucleotides of the primary DNA chain would be too weak, in the case of several triplets, to realize the recognition process. All possible AA replacements were tested for strength of interaction in the Z₁-Z₃ positions. Domains with fewer than 2 hydrogen bonds on the primary strand were considered to be unstable.

Table 2 presents the ZF AA triplets having the highest affinity for interaction with corresponding DNA triplets. These ZF triplets contain only the main residues presented in Column A of Table 1. Table 2 also presents the binding energy components (H-bonds, water bridges, van der Waals interactions) maintaining the ZF-DNA recognition process in specific contact regions.

As can be seen from Table 2, the participation of the complementary DNA strand in the process of ZF binding, combined with the number of interactions (H-bonds, water bridges and van der Waals interactions) possible in the three contact regions, when optimal combinations are used, makes it possible to show that a complex formation with all 64 DNA triplets can be achieved. Table 2 shows that the maximal number of H-bonds, the strongest of the three types of interactions, is obtained when the first nucleotide of the triplet is guanine or adenine.

In nucleotide triplets wherein the number of H-bonds possible is less than maximal, the deficiency is often partially compensated by a significant amount of water-bridging between critical AA's and the sugar-phosphate backbone.

Even in cases wherein the first nucleotide of the triplet is thymine, and the number of the H-bonds is lowest, 1) the formation of two ES-bonds between the AA in the Z₃ position, and the adenine and complementary thymine in the third contact position, and 2) probably, a single H-bond between thymine and serine or threonine in the second contact position, means that even TTN triplets can bind a ZF protein with sufficient affinity.

In any event, to obtain DBP's of the greatest effectiveness, attention should be paid to having the strongest interactions in the flanking contact points (1 and 3). If weaker combinations must be used, they would have less effect if positioned in the center contact point (2). It is important to note, however, that even weak binding in the contact points is important for establishing specificity.

Table 3 presents the main ZF AA triplets of Table 2, as well as the alternative AA's (shown in Column B of Table 1) which would be also expected to provide effective binding to the respective bases of a given DNA triplet. Table 3 also presents the binding energy components (H-bonds, water bridges, van der Waals interactions) maintaining the ZF-DNA recognition process in specific contact regions.

TABLE 1-Z1 Z1 Z1 Hydrogen Water Hydrophobic Codon Column A Column B Bonds Contacts Contacts AAC Q= E*/R₁-K₁-N₁=/D₁*/(H-/Y-/S-/T-) 6 0 0 AAG Q= E*/R-/K- 6 0 0 AAT Q= R-/K-/E* 6 0 0 ACC Q= E*/K₁- 6 0 0 ACT Q= E-/R₁-/K₁- 6 0 0 GAA R= K-/H₁-/Y₁-/Q₁- 6 0 0 GAC R= K-/H₁-/Y₁- 6 0 0 GAG R= H-/K-/Y-/Q- 6 0 0 GAT R= H-/K-/Y-/Q* 6 0 0 CCC R= H-/K-/Q-/N-/(Y-/S-/T-) 0 0 GCT R= H-/K-/Y-/Q- 6 0 0 ACA Q= R-/K-/N-/E-/D- 5 1 0 ACG Q= R-/K-/N-/E*/D- 5 1 0 AGA Q= E*/R₁-/K₁- 5 1 0 AGG Q= E*/R₁-/K₁- 5 1 0 CAA E* Q*/N₁*/D₁*/R₂-/K₂-/Y₂-/S₂-/T₂- 5 1 0 CAG E* Q*/N*/D*/R₂-/K₂- 5 1 0 CAT E* Q*/R₂-/K₂-/(D*/N*/S-/T-/Y₂-) 5 1 0 CCC E* Q*/N₁*/D₁*/R₂=/K₂-/Q₂*/N₂* 5 1 0 CCT E* Q*/R₂-/K₂- 5 1 0 GCA R= K-/Q-/(H-/Y-/N-/S-/T-) 5 1 0 CCG R= H-/K-/Y-/Q-/N-/S-/T- 5 1 0 CGA R= H-/K-/Q*/N*/Y₁- 5 1 AAA R-/K- 5 0 0 AGC Q= R-/K-/E* 5 0 0 AGT Q= E*/R₁-/K₁- 5 0 0 GGG R= K-/Q*/(H-/Y-/N-) 5 0 0 GGG R= H-/K-/Y-/Q*N-) 5 0 0 GGT R= H-/K-/Y-/Q-/N- 5 0 0 CAC E* Q*/R₂-/K₂- 4 2 0 CCA E* Q*/R₂-/K₂- 4 2 0 CCG E* Q*/N*/D*/R₂=/K₂- 4 2 0 CGA E* Q*/N*/D*/R₂=/K₂- 4 2 0 CGC E* Q*/N*/D*/R₂-/K₂-/Y₂-/Q₂-/(S-/T-) 4 1 0 CGG E* Q*/N*/D*/R₂-/K₂-/Q₃= 4 1 0 CGT E* Q*/D₁*/R₂=/K₂-/Q₂- 4 1 0 CTA E* Q* 3 1 1 CTC E* Q*/N₁-/D₁-/R₂=/K₂-/Q₂-/N₂- 3 1 1 CTG E* Q*/N*/D*/R₂-/K₂- 3 1 1 CTT E* Q*/N*/D*/R₂-/K₂-/Q₃= 3 1 1 TCA I#/L# R-/K-/Q* 3 1 1 TCG I#/L# R-/K-/Q* 3 1 1 TGA I#/L# R-/K-/Q* 3 1 1 TAC I#/L#/V# R-/K-/Q* 3 0 1 TGC I#/L₁#/V₁# R-/K-/H₁-/Q₁*/N₁*/S₁-/T₁- 3 0 1 TCG I#/L# R-/K-/Q* 3 0 1 TGT I# R-/H-/K-/Q*/N*/L# 3 0 1 TTA I#/L# R-/K-/Q*/N* 2 0 2 TTC I#/L#/V# R-/K-/Q*/N* 2 0 2 TTG I#/L# R-/K-/Y-/Q* 2 0 2 TTT I#/L# R-/K-/Q*/N*/V₁# 2 0 2 ATA Q= E*/R₁-/K₁- 4 0 1 ATC Q= N-/E*/D*/R₁-/K₁- 4 0 1 ATG Q= R-/K-/E-/(H-) 4 0 1 ATT Q= E*/R₁-/K₁- 4 0 1 GTA R= H-/K-/Y-/Q- 4 0 1 GTC R= H-/K-/Y-/Q* 4 0 1 GTG R= K-/Q-/H₁- 4 0 1 GTT R= H-/K-/Q-/N-/Y₁- 4 0 1 TAA I#/L# R-/K-/Q-/V₁# 4 0 1 TAG I#/L# R-/K-/Q- 4 0 1 TAT I#/L#/V# R-/K-/Y-/Q-/N- 4 0 1 TCC I#/L# R-/H-/K-/Q*/N*/V#/A# 4 0 1 TCT I₁#/L₁# R-/H-(K-/Q* 4 0 1 where / separates alternative amino acids where X without subscript has all its interactions with the primary strand where X₁ has some interactions with the primary strand and some interactions with the complementary strand where X₂ has interaction with the complementary strand where X₃ has interactions with both the primary and complementary strands where - is one hydrogen bond between the animo acid and the base where = is two hydrogen bonds between the amino acid and the base where is one hydrogen bond via a water bridge between the amino acid and the phosphodiester oxygen atom of the backbone where # is one or more van der Waals contacts between the amino acid and the base where amino acids in ( ) have interaction with the base of the primary strand where one of two other possible protein-DNA recognition interactions is absent

TABLE 1-Z2 CGC H₁- Q*/N*/S-/T-/K₁-/(R₁=/Y-) 4 1 0 CGG H- K-/Q*/N*/R₁-/(Y-) 4 1 0 CCT H- Q*/N*/R₁-/K₁-/(Y-) 4 1 0 ATA I#/L#/V#/A# S-/T-/K₁-/Q₁*/N₁*/(R-/H-/Y-) 4 0 1 ATC I#/L# N*/S-/T-/V-/R₁-/H₁-/K₁-/Y₁-/Q₁*/R₂-/K₂-/Q₂=/N₂=/E₂*/D₂* 4 0 1 ATG I#/L#/V# S-/T-/E₂-/D₂-/Q₃=/N₂=/(R-/H-/K-/Y-) 4 0 1 ATT I#/L#/V# R-/H-/K-/Y-/Q*/N*/S-/T- 4 0 1 GTA I#/L#/V#/A# Q*/N*/S-/T-/R₂-/H₁-/K₁-/E₂*/D₂*/Q₃=/N₃=/(Y-) 4 0 1 GTC I#/L# N-/S-/T-/V-/R₁-/H₁-/K₁-/Q₁* 4 0 1 GTG I#/L#/V# Q*/N*/S-/T-/H₁-/K₁- 4 0 1 GTT I#/L#/V# Q*/N*/S-/T-/K₁-/(R-/H-/Y-/A-) 4 0 1 TAA N= D*/R₁-/H₁-/K₁-/Y₁-/Q₁=/E₁- 4 0 1 TAG N= Q=/E*/D*/R₁-/K₁-/K₂- 4 0 1 TAT N= K-/Q=/N=/E*/D*/S-/T-/H₂-/H₂-/K₂-/Q₃=/N₃=/(R-/Y-) 4 0 1 TCC Q₃=/E* Q*/N*/D*/S-/T-/R₂-/H₂-/K₂/Y₂-/Q₂-/N₂- 4 0 1 TCT N₃=/D* Q*/N*/D*/S-/T-/R₂-/H₂-/K₂/Y₂-/Q₂* 4 0 1 CTA I#/L#/V#/A# S-/T-/Q₁=/N₁=(H-/K-) 3 1 1 CTC I#/L# S-/T-/V-/R₁-/H₁-/K₁-/Y₁-/Q₁*/N₁* 3 1 1 CTG I#/L#/V# N*/S-/T-/K₁-/Q₁* 3 1 1 CTT I#/L# Q*/N*/S-/T-/V#/R₁-/H₁-/K₁-/E₂-/D₂-/Q₂=/N₃=/(Y-) 3 1 1 TCA D* Q*/N*/E*/S-/T-/R₂=/H₂-/K₂-/Y₂-/Q₃=(N₂= 3 1 1 TCG D* Q*/N*/E*/S-/T-/R₂=/H₂-/K₂-/Y₂-/S₂-/T₂-/Q₃=(N₂= 3 1 1 TGA N* Q*/S-/T-/H₁-/K₁-/(R-/Y-) 3 1 1 TAC N= D-/H₁-/K₁-/Q₁=/E₁*/k₂/(R-/Y-) 3 0 1 TGC H₁- Q*/N*/S-/T-/R₁=/K₁-/Y₁- 3 0 1 TGG H- R-/K-/Q*/N*/Y₁- 3 0 1 TGT H₁- N*/S-/T-/K₁-/Y₁-/Q₁*/(R=) 3 0 1 TTA I#/L#/V#/A# S-/T-/R₁-/H₁/K₁-/Y₁-/E₂-/D₂-/Q₃=/N₃= 2 0 2 TTC I#/L# N*/S-/T-V-/A-/R₁/H₁-/K₁-/Y₁-/K₂-/Q₃=/N₃= 2 0 2 TTG I#/L#/V# N*/S-/T-/H₁-/K₁-/Q₁* 2 0 2 TTT I#/L#/V# Q*/N*/S-/T-/A-/H₁-/K₁-/(R-/Y-) 2 0 2 AAC Q₁= N=/D*/S-/T-/R₁-/K₁-/E₁*/(H-/Y-) 6 0 0 AAG Q=/N= R-/H-/K-/E*/D*/K₂0 6 0 0 AAT N= K-/Q=/E*/D*/R₁-/H_(Q)-/K₂-/Q₃=/N₃= 6 0 0 ACC Q₃=/E* D*/S-/T-/N₃=/(K₂-) 6 0 0 ACT N₃=/D* Q*/N*/E*/S-/T-/K₂-/Q₃= 6 0 0 GAA N= D*/R₁-/H₁-/K₁-/Y₁/Q₁=/E₁*/K₂- 6 0 0 GAC N= D*/R₁-/H₁-/K₁-/Y₁/Q₁=/E₁*/K₂- 6 0 0 GAG N= Q=/E*/D*/R₁-/H₁-/K₁-/7₁-/K₂- 6 0 0 GAT Q=/N= K-/E*/D*/K₂-/(R-/H-/Y-) 6 0 0 GCC Q₃=/E* Q*/N*/D*/S-/T-R₂/H₂-/K₂-/Q₂-/N₂-/S₂-/T₂- 6 0 0 GCT N₃=/D* Q-/N-/E-/S-/T-/H₂-/K₂-/N₂*/Q₃=/(R₂=/Y₂-) 6 0 0 ACA D* Q*/N*/E*/S-/T-K₂- 5 1 0 ACG E*/D* Q*/N*/S-/T-/R₂-/H₂/K₂-/Y₂-/Q₃=/N₃= 5 1 0 AGA N* R₁-/H₁-/K₁-/Y₁-/Q₁* 5 1 0 AGG Q*/N* R₁*/H₁*/K₁- 5 1 0 CAA N= D*/S-/T-/R₁-/H₁-/K₁-/Y₁-/Q₁=/E₁* 5 1 0 CAG Q= N=/E*/D*/R₁-/K₁-/K₂-/Q₃= 5 1 0 CAT Q₁=/N= D-/S-/T-/R₁-/H₁-/K₁-/Y₁-/E₁=/K₂-/Q₃=N₃= 5 1 0 CCC Q₂=/E₁* N*/D*/S-/T-/H₂-/K₂-/N₃=/(Y₂-) 5 1 0 CCT N₃=/D* Q*/N*/E*/S-/T-/H₂-/K₂-/Y_(A)-/Q₃=/N₂= 5 1 0 GCA D* Q*/N*/E*/S-/T-/R₂-/H₂-/K₂-/Y₂-/Q₃=/N₃= 5 1 0 GCG E*/D* Q*/N*/S-/T-/K₂-/Q₃=/N₃=/(H₂-) 5 1 0 GGA N* Q*/S-/T-/K₁-/(R-/Y-/H-) 5 1 0 AAA N= D*/R₁/H₁/K₁/Y₁/Q₁=/E₁* 5 0 0 AGC H₁- Q*/N*/S-/T-/R₁-/K₁-/(R=/Y-) 5 0 0 AGT H₁- Q*/N*/S-/T-/R₁-/K₁- 5 0 0 GGC H₁- N*/S-/T-/K₁-/(R-/K-/7-/Q*) 5 0 0 GGG H- K-/Q*/N*/S-/T-/Y₁-/(R-) 5 0 0 GGT H- Q*/N*/S-/T-/R₁-/K₁- 5 0 0 CAC N= D*/R₁-/K₁-/Q₁=/E₁* 4 2 0 CCA D* N*/S-/T-/Q₁*/E₁*/K₂-/N₃= 4 2 0 CCG D* Q*/N*/E*/S-/T-/K₂-/Q₃=/N₃/(R-/H-/Y-) 4 2 0 CGA N* Q*/S-/T-/R₁=/H₁-/K₁-/(Y-) 4 2 0 where / separates alternative amino acids where X without subscript has all its interactions with the primary strand where X₁ has some interactions with the primary strand and some interactions with the complementary strand where X₂ has interaction with the complementary strand where X₃ has interactions with both the primary and complementary strands where - is one hydrogen bond between the animo acid and the base where = is two hydrogen bonds between the amino acid and the base where is one hydrogen bond via a water bridge between the amino acid and the phosphodiester oxygen atom of the backbone where # is one or more van der Waals contacts between the amino acid and the base where amino acids in ( ) have interaction with the base of the primary strand where one of two other possible protein-DNA recognition interactions is absent

TABLE 1-Z3 Z3 Z3 Hydrogen Water Hydrophobic Codon Column A Column B Bonds Contacts Contacts AAC Q= E*/R₁-K₁-N₁=/D₁*/(H-/Y-/S-/T-) 6 0 0 AAG Q= E*/R-/K- 6 0 0 AAT Q= R-/K-/E* 6 0 0 ACC Q= E*/K₁- 6 0 0 ACT Q= E-/R₁-/K₁- 6 0 0 GAA R= K-/H₁-/Y₁-/Q₁- 6 0 0 GAC R= K-/H₁-/Y₁- 6 0 0 GAG R= H-/K-/Y-/Q- 6 0 0 GAT R= H-/K-/Y-/Q* 6 0 0 CCC R= H-/K-/Q-/N-/(Y-/S-/T-) 0 0 GCT R= H-/K-/Y-/Q- 6 0 0 ACA Q= R-/K-/N-/E-/D- 5 1 0 ACG Q= R-/K-/N-/E*/D- 5 1 0 AGA Q= E*/R₁-/K₁- 5 1 0 AGG Q= E*/R₁-/K₁- 5 1 0 CAA E* Q*/N₁*/D₁*/R₂-/K₂-/Y₂-/S₂-/T₂- 5 1 0 CAG E* Q*/N*/D*/R₂-/K₂- 5 1 0 CAT E* Q*/R₂-/K₂-/(D*/N*/S-/T-/Y₂-) 5 1 0 CCC E* Q*/N₁*/D₁*/R₂=/K₂-/Q₂*/N₂* 5 1 0 CCT E* Q*/R₂-/K₂- 5 1 0 GCA R= K-/Q-/(H-/Y-/N-/S-/T-) 5 1 0 CCG R= H-/K-/Y-/Q-/N-/S-/T- 5 1 0 CGA R= H-/K-/Q*/N*/Y₁- 5 1 AAA R-/K- 5 0 0 AGC Q= R-/K-/E* 5 0 0 AGT Q= E*/R₁-/K₁- 5 0 0 GGG R= K-/Q*/(H-/Y-/N-) 5 0 0 GGG R= H-/K-/Y-/Q*N-) 5 0 0 GGT R= H-/K-/Y-/Q-/N- 5 0 0 CAC E* Q*/R₂-/K₂- 4 2 0 CCA E* Q*/R₂-/K₂- 4 2 0 CCG E* Q*/N*/D*/R₂=/K₂- 4 2 0 CGA E* Q*/N*/D*/R₂=/K₂- 4 2 0 CGC E* Q*/N*/D*/R₂-/K₂-/Y₂-/Q₂-/(S-/T-) 4 1 0 CGG E* Q*/N*/D*/R₂-/K₂-/Q₃= 4 1 0 CGT E* Q*/D₁*/R₂=/K₂-/Q₂- 4 1 0 CTA E* Q* 3 1 1 CTC E* Q*/N₁-/D₁-/R₂=/K₂-/Q₂-/N₂- 3 1 1 CTG E* Q*/N*/D*/R₂-/K₂- 3 1 1 CTT E* Q*/N*/D*/R₂-/K₂-/Q₃= 3 1 1 TCA I#/L# R-/K-/Q* 3 1 1 TCG I#/L# R-/K-/Q* 3 1 1 TGA I#/L# R-/K-/Q* 3 1 1 TAC I#/L#/V# R-/K-/Q* 3 0 1 TGC I#/L₁#/V₁# R-/K-/H₁-/Q₁*/N₁*/S₁-/T₁- 3 0 1 TCG I#/L# R-/K-/Q* 3 0 1 TGT I# R-/H-/K-/Q*/N*/L# 3 0 1 TTA I#/L# R-/K-/Q*/N* 2 0 2 TTC I#/L#/V# R-/K-/Q*/N* 2 0 2 TTG I#/L# R-/K-/Y-/Q* 2 0 2 TTT I#/L# R-/K-/Q*/N*/V₁# 2 0 2 ATA Q= E*/R₁-/K₁- 4 0 1 ATC Q= N-/E*/D*/R₁-/K₁- 4 0 1 ATG Q= R-/K-/E-/(H-) 4 0 1 ATT Q= E*/R₁-/K₁- 4 0 1 GTA R= H-/K-/Y-/Q- 4 0 1 GTC R= H-/K-/Y-/Q* 4 0 1 GTG R= K-/Q-/H₁- 4 0 1 GTT R= H-/K-/Q-/N-/Y₁- 4 0 1 TAA I#/L# R-/K-/Q-/V₁# 4 0 1 TAG I#/L# R-/K-/Q- 4 0 1 TAT I#/L#/V# R-/K-/Y-/Q-/N- 4 0 1 TCC I#/L# R-/H-/K-/Q*/N*/V#/A# 4 0 1 TCT I₁#/L₁# R-/H-(K-/Q* 4 0 1 where / separates alternative amino acids where X without subscript has all its interactions with the primary strand where X₁ has some interactions with the primary strand and some interactions with the complementary strand where X₂ has interaction with the complementary strand where X₃ has interactions with both the primary and complementary strands where - is one hydrogen bond between the animo acid and the base where = is two hydrogen bonds between the amino acid and the base where is one hydrogen bond via a water bridge between the amino acid and the phosphodiester oxygen atom of the backbone where # is one or more van der Waals contacts between the amino acid and the base where amino acids in ( ) have interaction with the base of the primary strand where one of two other possible protein-DNA recognition interactions is absent

TABLE 2 Z1 Z2 Z3 Hydrogen Water Hydrophobic Codon Column A Column A Column A Bonds Contacts Contacts AAC Q Q R 6 0 0 AAG Q N/Q R 6 0 0 AAT Q N Q 6 0 0 ACC Q E/Q R 6 0 0 ACT Q D/N Q 6 0 0 GAA R N Q 6 0 0 GAC R N E/Q 6 0 0 GAG R N R 6 0 0 GAT R N/Q Q 6 0 0 GCC R E/Q R 6 0 0 GCT R D/N Q 6 0 0 ACA Q D Q 5 1 0 ACG Q D/E R 5 1 0 AGA Q N Q 5 1 0 AGG Q N/Q R 5 1 0 CAA E N Q 5 1 0 CAG E Q R 5 1 0 CAT E N/Q N/Q 5 1 0 CCC E E/Q R 5 1 0 CCT E D/N Q 5 1 0 GCA R D Q 5 1 0 GCG R D/E R 5 1 0 GGA R N Q 5 1 0 AAA K/R N Q 5 0 0 AGC Q H R 5 0 0 AGT Q H Q 5 0 0 GGC R H R 5 0 0 GGG R H R 5 0 0 GGT R H N/Q 5 0 0 CAC E N E 4 2 0 CCA E D Q 4 2 0 CCG E D R 4 2 0 CGA E N Q 4 2 0 CGC E H R 4 1 0 CGG E H R 4 1 0 CGT E H N/Q 4 1 0 ATA Q A/I/L/V Q 4 0 1 ATC Q I/L E/R 4 0 1 ATG Q I/L/V R 4 0 1 ATT Q I/L/V Q 4 0 1 GTA R A/I/L/V Q 4 0 1 GTC R I/L E/R 4 0 1 GTG R I/L/V R 4 0 1 GTT R I/L/V Q 4 0 1 TAA I/L N Q 4 0 1 TAG I/L N R 4 0 1 TAT I/L/V N Q 4 0 1 TCC I/L E/Q R 4 0 1 TCT I/L D/N N/Q 4 0 1 CTA E A/I/L/V Q 3 1 1 CTC E I/L E/R 3 1 1 CTG E I/L/V R 3 1 1 CTT E I/L Q 3 1 1 TCA I/L D Q 3 1 1 TCG I/L D R 3 1 1 TGA I/L N Q 3 1 1 TAC I/L/V N E 3 0 1 TGC I/L/V H R 3 0 1 TGG I/L H R 3 0 1 TGT I H Q 3 0 1 TTA I/L A/I/L/V Q 2 0 2 TTC I/L/V I/L E/R 2 0 2 TTG I/L I/L/V R 2 0 2 TTT I/L I/L/V Q 2 0 2 where / separates alternative amino acids

TABLE 3 Z1 Z2 Z3 Hydro- Water Hydro- Co- Column Z1 Column Z2 Column Z3 gen Con- phobic don A Column B A Column B A Column B Bonds tacts Contacts AAC Q D/E/H/K/N/R/S/T/Y Q D/E/H/K/N/R/S/T/Y R D/E/H/K/N/Q/Y 6 0 0 AAG Q E/K/R N/Q D/E/H/K/R R D/E/H/K/N/Q/S/T/Y 6 0 0 AAT Q E/K/R N D/E/H/K/N/Q/R Q D/E/H/K/N/Q/R/Y 6 0 0 ACC Q E/K E/Q D/K/N/S/T R E/K/N/Q/S/T 6 0 0 ACT Q E/K/R D/N E/K/N/Q/S/T Q D/E/H/K/N/Q/R/Y 6 0 0 GAA R H/K/Q/Y N D/E/H/K/Q/R/Y Q E/H/K/Q/R/Y 6 0 0 GAC R H/K/Y N D/E/H/K/Q/R/Y E/Q H/K/N/Q/R/S/T/Y 6 0 0 GAG R H/K/Q/Y N D/E/H/K/Q/R/Y R D/E/K/N/Q/S/T 6 0 0 GAT R H/K/Q/Y N/Q D/E/H/K/R/Y Q D/E/H/I/K/N/Q/R/Y 6 0 0 GCC R H/K/N/Q/S/T/Y E/Q D/H/K/N/Q/R/S/T R D/E/H/K/N/Q/S/T/Y 6 0 0 GCT R H/K/Q/Y D/N E/H/K/N/Q/R/S/T/Y Q D/E/H/K/N/Q/R/S/T/Y 6 0 0 ACA Q D/E/K/N/R D E/K/N/Q/S/T o D/E/H/I/K/L/N/Q/R/S/T/V/Y 5 1 0 ACG Q D/E/K/N/R D/E H/K/N/Q/R/S/T/Y R D/E/H/K/N/Q/S/T/Y 5 1 0 AGA Q E/K/R N H/K/Q/R/Y Q A/E/H/I/K/L/N/Q/R/V/Y 5 1 0 AGG Q E/K/R N/Q H/K/R R D/E/K/N/Q/Y 5 1 0 CAA E D/K/N/Q/R/S/T/Y N DfE/H/K/Q/R/S/T/Y Q E/H/K/Q/R/Y 5 1 0 CAG E D/K/N/Q/R Q D/E/K/N/Q/R R D/E/H/K/N/Q/S/T/Y 5 1 0 CAT E D/K/N/Q/R/S/T/Y N/Q D/E/H/K/N/Q/R/S/T/Y N/Q D/E/H/K/Q/R/Y 5 1 0 CCC E D/K/N/Q/R E/Q D/H/K/N/S/T/Y R E/H/KfN/Q/S/T/Y 5 1 0 CCT E K/Q/R D/N E/H/K/N/Q/S/T Q E/H/K/N/Q/R/S/T/Y 5 1 0 GCA R H/K/N/Q/S/T/Y D E/H/K/N/Q/R/S/T/Y Q A/E/H/I/K/L/N/Q/R/S/T/V/Y 5 1 0 GCG R H/K/N/Q/S/T/Y D/E H/K/N/Q/S/T R D/E/H/K/N/Q/S/T/Y 5 1 0 GGA R H/K/N/Q/Y N H/K/Q/R/S/T/Y Q A/D/E/H/I/K/L/N/Q/R/V/Y 5 1 0 AAA K/R N D/E/H/K/Q/R/Y Q D/E/H/I/K/L/N/Q/R/Y 5 0 0 AGC Q E/K/R H K/N/Q/R/S/T/Y R D/E/H/K/N/Q/Y 5 0 0 AGT Q E/K/R H K/N/Q/R/S/T Q D/E/H/I/K/L/N/Q/R/S/T/Y 5 0 0 GGC R H/K/N/Q/Y H K/N/Q/R/S/T/Y R D/E/H/K/N/Q/S/T/Y 5 0 0 GGG R H/K/N/Q/Y H K/N/Q/R/S/T/Y R D/E/H/K/N/Q/S/T/Y 5 0 0 GGT R H/K/N/Q/Y H K/N/Q/R/S/T N/Q D/E/K/N/Q/R/S/T 5 0 0 CAC E K/Q/R N D/E/K/Q/R E H/K/Q/R/Y 4 2 0 CCA E K/Q/R D E/K/N/Q/S/T Q A/E/H/I/K/L/N/Q/R/S/T/V/Y 4 2 0 CCG E D/K/N/Q/R D E/H/K/N/Q/R/S/T/Y R D/E/H/K/N/Q 4 2 0 CGA E D/K/N/Q/R N H/K/Q/R/S/T/Y Q A/D/E/H/I/K/L/N/Q/R/S/T/V/Y 4 2 0 CGC E D/K/N/Q/R/S/T/Y H K/N/Q/R/S/T/Y R D/E/H/K/N/Q/Y 4 1 0 CGG E D/K/N/Q/R H K/N/Q/R/Y R D/E/H/K/N/Q/S/T/Y 4 1 0 CGT E D/K/Q/R H K/N/Q/R/Y N/Q D/E/H/K/N/Q/R/Y 4 1 0 ATA Q E/K/R A/I/L/V H/K/N/Q/R/S/T/Y Q E/H/I/K/L/O/R/V/Y 4 0 1 ATC Q D/E/K/N/R I/L D/E/H/K/N/Q/R/S/T/V/Y E/R H/K/N/Q/R/Y 4 0 1 ATG Q E/H/K/R I/L/V D/E/H/K/N/Q/R/S/T/Y R D/E/H/K/N/Q/Y 4 0 1 ATT Q E/K/R I/L/V H/K/N/Q/R/S/T/Y Q D/E/H/K/N/Q/R/S/T/Y 4 0 1 GTA R H/K/Q/Y A/I/L/V D/E/H/K/N/Q/R/S/T/Y Q A/D/E/H/I/K/L/N/Q/R/S/T/V/Y 4 0 1 GTC R H/K/Q/Y I/L H/K/N/Q/R/S/T/V E/R H/K/N/Q/S/T/Y 4 0 1 GTG R H/K/Q E/L/V H/K/N/Q/S/T R D/E/H/K/N/Q/S/T/Y 4 0 1 GTT R H/K/N/Q/Y I/L/V A/H/K/N/Q/R/S/T/Y Q D/E/H/I/K/L/N/Q/R/V/Y 4 0 1 TAA I/L K/O/R/V N D/E/H/K/Q/R/Y Q A/E/H/I/K/L/Q/R/V/Y 4 0 1 TAG I/L K/Q/R N D/E/K/Q/R R D/E/H/K/N/O/Y 4 0 1 TAT E/L/V K/N/Q/R/Y N D/E/H/K/N/Q/R/S/T/Y Q D/E/H/I/K/L/N/O/R/S/T/V/Y 4 0 1 TCC E/L A/H/*K/N/Q/R/V E/Q D/H/K/N/Q/R/S/T/Y R E/H/K/N/Q/S/T/Y 4 0 1 TCT I/L H(K/Q/R D/N E/H/K/N/Q/R/S/T/Y N/Q D/E/H/K/Q/R/Y 4 0 1 CTA E Q A/I/L/V H/K/N/Q/S/T Q A/E/I/K/L/N/Q/R/S/T/V 3 1 1 CTC E D/K/N/Q/R I/L H/K/N/Q/R/S/T/V/Y E/R D/H/*K/N/Q/Y 3 1 1 CTG E D/K/N/Q/R I/L/V K/N/Q/S/T R D/E/K/N/Q/Y 3 1 1 CTT E D/K/N/Q/R I/L D/E/H/K/N/Q/R/S/T/V/Y Q D/E/H/K/N/Q/R/Y 3 1 1 TCA I/L K/Q/R D E/H/K/N/Q/R/S/T/Y Q A/E/H/I/K/L/N/Q/R/S/T/V/Y 3 1 1 TCG E/L K/O/R D E/H/K/N/Q/R/S/T/Y R D/E/H/K/N/Q/S/T/Y 3 1 1 TGA E/L K(Q/R N H/K/Q/R/S/T/Y Q D/E/H/K/N1O/R/Y 3 1 1 TAC I/L/V K/Q/R N D/E/H/K/Q/R/Y E H/K/N/Q/R/Y 3 0 1 TGC E/L/V H/K/N/Q/R/S/T H K/N/Q/R/S/T/Y R D/E/H/K/N/O/Y 3 0 1 TGG I/L K/Q/R H K/N/Q/R/Y R D/E/H/K/N/Q/S/T/Y 3 0 1 TGT I H/K/L/N/Q/R H K/N/Q/R/S/T/Y Q E/H/E/K/L/N/Q/R/Y 3 0 1 TTA I/L K/N/Q/R A/I/L/V D/E/H/K/N/O/R/S/T/Y Q A/E/H/I/K/L/N/R/S/T/V/Y 2 0 2 TTC E/L/V K/N/Q/R I/L A/H/K/N/Q/R/S/T/V/Y E/R D/H/K/N/Q/S/T/Y 2 0 2 TTG I/L K/Q/R/Y I/L/V H/K/N/Q/S/T R D/E/H/K/N/Q/Y 2 0 2 TTT I/L K/N/Q/R/V I/L/V A/H/K/N/Q/R/S/T/Y Q D/E/H/K/N/Q/R/Y 2 0 2 where / separates alternative amino acids

The results of the molecular modeling analysis of various ZF a-helix complexes with the 64 different DNA triplets (Tables 1, 2 and 3), and the findings of spatial peculiarities in the three contact positions, are reflected in the ZF-DNA recognition rules. On the basis of the rules set forth in Tables 1, 2 and 3, DBP's with optimal binding affinity for any target DNA sequence can be designed. The “Column A” designations, i.e., the “A Rules,” in Tables 1-3, show the amino acids with optimal binding for a given codon (triplet). The “Column B” designations, i.e., the “B rules,” in Tables 1 and 3, show the amino acids with secondary, but still significant, binding affinity for a given triplet.

The column A rules range from the strongest triplet recognition with six H-bonds, zero water contracts and zero hydrophobic contacts with an evaluated energy of (5×6)+(2×0)+(×0)=30 to two hydrogen bonds, zero water contacts and two hydrophobic contacts with an evaluated energy of (5×2)+(2×0)+(1×2)=12. The Column A rules ordinarily have a choice of just one or two amino acids in positions Z₁, Z₂ and Z₃. The column B rules, by comparison, have from three possible amino acids in each of the Z₁, Z₂ and Z₃ positions to as many as eighteen amino acids in different contacting arrangements in each of the Z₁, Z₂ and Z₃ positions. In the evaluation of the column B energies, there are a large number different groupings of three amino acids in positions Z₁, Z₂ and Z₃. The minimum energy is three hydrogen bonds, zero water contacts and zero hydrophobic contacts with an evaluated energy of (5×3)+(2×0)+(1×0)=15. The maximum energy evaluation for these combinations is, on average, three hydrogen bonds and either two water contacts or two hydrophobic contacts, with an evaluated energy of from (5×3)+(2×2)+(1×0)=19 down to (5×3)+(2×0)+(1×2)=17. Thus, the column B rules have a narrower energy range (i.e., from 19 down to 15) than do the column A rules, which have an energy range from 30 down to 12. The narrow energy range for the column B rules means that the 64 different rules do not distinguish on the basis of energy as well as the 64 column A rules.

For example, as set forth in Table 2, a DBP which binds optimally to the DNA base triplet guanine-cytosine-cytosine (GCC) is one wherein the portion of the protein responsible for the binding to the triplet is a ZF domain within which is contained a segment having the sequence Z₃XXZ₂LXZ₁H (SEQ ID NO: 2), wherein Z₁ is an arginine which interacts with position 1 of the DNA triplet; Z₂ is a glutamine or a glutamic acid which interacts with position 2 of the DNA triplet; Z₃ is an arginine which interacts with position 3 of the DNA triplet; X is an arbitrary amino acid; L is leucine and H is histidine.

As set forth in Table 1 or 3 (see the “column B” entries for the Z₁, Z₂, and Z₃ positions for a given codon), a DBP which effectively, if not optimally, binds to the DNA base triplet guanine-cytosine-cytosine (GCC) is one wherein the portion of the protein responsible for the binding to the triplet is a ZF domain within which is contained a segment having the sequence Z₃XXZ₂LXZ₁H (SEQ ID NO: 2), wherein Z₁ is an amino acid selected from the group consisting of histidine, lysine, glutamine, asparagine, tyrosine, serine and threonine which interacts with position 1 of the DNA triplet; Z₂ is an amino acid selected from the group consisting of glutamine, asparagine, aspartic acid, serine, threonine, arginine, histidine, and lysine which interacts with position 2 of the DNA triplet; Z₃ is an amino acid selected from the group consisting of glutamine, asparagine, glutamic acid, aspartic acid, histidine, lysine, tyrosine, serine and threonine which interacts with position 3 of the DNA triplet; X is an arbitrary amino acid; L is leucine and H is histidine.

It will be appreciated, of course, that DBP's of intermediate affinity, i.e., ones wherein the Z₁, Z₂ and Z₃ contact amino acids are selected according to a combination of the “A” and “B Rules,” can be designed. For example, in the segment Z₃XXZ₂LXZ₁H (SEQ ID NO: 2) within a ZF domain for binding to the triplet GCC, Z₁ could be an arginine; Z₂ could be a glutamnine or a glutamic acid; and Z₃ could be selected from the group consisting of glutamine, asparagine, glutamic acid, aspartic acid, histidine, lysine, tyrosine, serine and threonine.

The basic building block for such proteins is denoted by the formula:

NH₂—ZiF_(c)—COOH,

where ZiF_(c) is a ZF domain of the form

Y/FXCX₂₋₄ CG/D K/RXFXZ₃XXZ₂LXZ₁HX₃₋₅ H   (sites 1-20 of SEQ ID NO: 1),

where

Z₁, Z₂ and Z₃ are amino acids chosen from Table 1, 2 or 3 to correspond to the three bases of the DNA triplet, and the remaining components of the formula are as described earlier in the description of Formula I.

In the preferred embodiment of the invention, a zinc-finger domain for binding to a given DNA triplet is designed by selection of the appropriate AA's in Table 2 or in column A of Table 1 or Table 3. In another embodiment of the invention, the ZF domain is designed by selection from among the AA's set forth for a given DNA triplet in column B of Table 1 or 3.

One such domain is required for each triplet of the target sequence; for a target string of only 3 bases, the above formula defines the protein.

If the target string of DNA is 6 bases, the DBP design is extended as follows:

NH₂—ZiF₁—{linker}—ZiF₂—COOH

where ZiF₁ and ZiF₂ are ZF domains designed, as shown above for ZiFC, to bind to the first and second triplets of the six bases, and (linker) is an amino acid sequence conforming to the pattern

T/S G/E X₀₋₂E K/R P   (sites 21-26 of SEQ ID NO: 1),

again wherein the components are as defined previously in Formula I.

If 1) the target string of DNA contains 9, 12, or a higher multiple of 3 bases; 2) it is required to design a DBP for 3n+3 bases; and 3) the DBP for the first 3n bases is given by the sequence:

NH₂—ZiF₁—{linker}—ZiF₂—{linker}— . . . —{linker}—ZiFn—COOH

then the DBP design is extended recursively and the required DBP is specified by the sequence:

NH₂—ZiF₁—{linker}—ZiF₂—{linker}— . . .

. . . —{linker}—ZiF_(n)—{linker}—ZiF_(n+1)——COOH

where ZiF_(n+1) is a ZF domain designed, as shown above for ZiFC, to bind with the n^(th)+1 triplet of the target sequence of base pairs.

FIG. 3 provides a schematic representation of a ZF protein wherein n=3, i.e., one which has 3 ZF domains (i.e., n=3) connected by linker sequences and is designed to bind to a target DNA string of 9 (3n) bases.

The above rules enable ready determination of the optimal amino acid(s) for binding to any given DNA triplet and thus the identification and positioning of the 3 amino acids in a ZF domain which would be the ideal component of a DBP for binding to the DNA triplet.

The application of the rules can then be extended to design of a DBP containing a set number, n_(d), of ZF domains, which DPB binds to a target stretch of 3n_(d) nucleotides within a given DNA sequence. The target 3n_(d) stretch of nucleotides, and the collection and order of n_(d) domains in the DBP, are such that the binding energy for the DPB and target DNA sequence is the highest possible for any pairing of a DBP containing the set number, n_(d), of ZF domains with any stretch of 3n_(d) nucleotides within the entire DNA molecule being screened.

Accordingly, the embodiment of the invention of primary importance is a method for designing such a DBP for a DNA sequence of any length. The method employs the rules disclosed above in combination with a means of screening and ranking all possible segments of 3n_(d) nucleotides within the sequence by their affinities for DBP's containing n_(d) ZF domains to determine a unique DBP with the desired properties.

More particularly, the invention is directed to a method for designing a DBP, with multiple ZF domains connected by linker sequences, that binds selectively to a target DNA sequence within a given gene, each of said ZF domains having the formula

A₁XCX₂₋₄CA₂A₃XFXZ₃XXZ₂LXZ₁HX₃₋₅H   (SEQ ID NO: 3)

and each of said linkers having the formula

A₄A₅X₀₋₂EA₆P   (SEQ ID NO: 4),

wherein

(i) X is any amino acid; (ii) X₂₋₄ is a peptide from 2 to 4 amino acids in length; (iii) X₃₋₅ is a peptide from 3 to 5 amino acids in length; (iv) X₀₋₂ is a peptide from 0 to 2 amino acids in length; (iv) A₁ is selected from the group consisting of phenylalanine and tyrosine; (v) A₂ is selected from the group consisting of glycine and aspartic acid; (vi) A₃ is selected from the group consisting of lysine and arginine; (vii) A₄ is selected from the group consisting of threonine and serine; (viii) A₅ is selected from the group consisting glycine and glutamic acid; (ix) A₆ is selected from the group consisting of lysine and arginine; (x) C is cysteine; (xi) F is phenylalanine; (xii) L is leucine; (xiii) H is histidine; (xiv) E is glutamic acid; (xv) P is proline; and (xvi) Z₁, Z₂ and Z₃ are the base-contacting amino acids, comprising the steps of:

(a) setting a genome to be screened;

(b) selecting the target DNA sequence in the genome for binding;

(c) setting the number of zinc-finger domains to n_(d);

(d) dividing the target DNA sequence into nucleotide blocks wherein each block contains n_(z) nucleotides using a first routine where n_(z) is determined using the following relationship:

n_(z)=3n_(d);

(e) assigning base-contacting amino acids at Z₁, Z₂ and Z₃ to each ZF domain, according to the A Rules and /or B Rules set forth in Tables 1-3, of a DBP which binds to the first nucleotide block from step (d) as numbered from the first 5′ nucleotide of the target gene sequence to generate a block-specific DBP and calculating the binding energy, Binding Energy block, of each ZF domain of each such block-specific DBP as the product of the binding energies, Binding Energy_(domain,) of all zinc-finger domains of the polypeptide, each determined using the formula:

Binding Energy_(domain,)=(5×the number of hydrogen bonds)+(2×the number of H₂O contacts)+(the number of hydrophobic contacts);

(f) subdividing the DBP from step (d) into blocks using a second routine to generate a subdivided DBP having three ZF domains;

(g) screening the subdivided DBP from step (f) against the genome using a third routine to determine the number of binding sites in the genome for each subdivided DBP in the genome and assigning a binding energy for each such site using the following formula:

Binding Energy_(site n)=(5×the number of hydrogen bonds)+(2×the number of H₂O contacts)+(the number of hydrophobic contacts);

(h) calculating a ratio of binding energy, R_(b), using a fourth routine for each nucleotide-block-specific DBP from step (e) using the following formula:

R_(b)=Binding Energy_(block)/the sum of all Binding Energy_(site n)'s for all subdivided DBP's from step (g);

(i) repeating steps (f) through (h) for each subdivided DBP wherein n_(d)≧4;

(j) repeating steps (d) through (i) for each nucleotide block in the target DNA sequence containing n_(z) nucleotides;

(k) rank-ordering R_(b) numerical values obtained from step (h); and

(l) selecting a DBP with an acceptable R_(b) value.

Preferred embodiments of this aspect of the invention are:

1) the design method as set forth above wherein the DBP R_(b) numerical value is the highest numerical value for all DBP's in step (h) that bind to the target DNA sequence.

2) the method above wherein the DBP R_(b) numerical value determined in step (h) is at least 10,000.

3) the method above wherein the number of ZF domains, n_(d), is nine.

4) the method above wherein the rules for assigning base-contacting amino acids at Z₁, Z₂ and Z₃ for each nucleotide block in step (e) are selected from rule set A.

The invention is further directed to a computer system for designing a DBP, with multiple ZF domains connected by linker sequences, that binds selectively to a target DNA sequence within a given gene, each of said ZF domains having the formula

A₁XCX₂₋₄CA₂A₃XFXZ₃XXZ₂LXZ₁HX₃₋₅H   (SEQ ID NO: 3)

and each of said linkers having the formula

A₄A₅X₀₋₂EA₆P   (SEQ ID NO: 4),

wherein

(i) X is any amino acid; (ii) X₂₋₄ is a peptide from 2 to 4 amino acids in length; (iii) X₃₋₅ is a peptide from 3 to 5 amino acids in length; (iv) X₀₋₂ is a peptide from 0 to 2 amino acids in length; (iv) A₁ is selected from the group consisting of phenylalanine and tyrosine; (v) A₂ is selected from the group consisting of glycine and aspartic acid; (vi) A₃ is selected from the group consisting of lysine and arginine; (vii) A₄ is selected from the group consisting of threonine and serine; (viii) A₅ is selected from the group consisting glycine and glutamic acid; (ix) A₆ is selected from the group consisting of lysine and arginine, (x) C is cysteine; (xi) F is phenylalanine; (xii) L is leucine; (xiii) H is histidine; (xiv) E is glutamic acid; (xv) P is proline; and (xvi) Z₁, Z₂ and Z₃ are the base-contacting amino acids, comprising the steps of:

(a) setting a genome to be screened;

(b) selecting the target DNA sequence in the genome for binding;

(c) setting the number of ZF finger domains to n_(d);

(d) dividing the target DNA sequence into nucleotide blocks wherein each block contains n_(z) nucleotides using a first routine where n_(z) is determined using the following relationship:

n_(z)=3n_(d);

(e) assigning base-contacting amino acids at Z₁, Z₂ and Z₃ to each ZF domain, according to the A Rules and/or B Rules set forth in Tables 1-3, of a DBP which binds to the first nucleotide block from step (d) as numbered from the first 5′ nucleotide of the target gene sequence to generate a block-specific DBP and calculating the binding energy, Binding Energy_(block,) of each ZF domain of each such block-specific DBP as the product of the binding energies, Binding Energy_(domain,) of all domains of the DBP, each determined using the formula:

Binding Energy_(domain)=(5×the number of hydrogen bonds)+(2×the number of H₂O contacts)+(the number of hydrophobic contacts);

(f) subdividing the DBP from step (d) into blocks using a second routine to generate a subdivided DBP having three ZF domains;

(g) screening the subdivided DBP from step (f) against the genome using a third routine to determine the number of binding sites in the genome for each subdivided DBP in the genome and assigning a binding energy for each such site using the following formula:

Binding Energy_(site n)=(5×the number of hydrogen bonds)+(2×the number of H₂O contacts)+(the number of hydrophobic contacts);

(h) calculating a ratio of binding energy, R_(b), using a fourth routine for each nucleotide block-specific DBP from step (e) using the following formula:

R_(b)=Binding Energy_(block)/the sum of all Binding Energy_(site n)'S for all subdivided DBP's from step (g);

(i) repeating steps (f) through (h) for each subdivided DBP wherein n_(d)≧4;

(j) repeating steps (d) through (i) for each nucleotide block in the target DNA sequence containing n_(z) nucleotides;

(k) rank-ordering R_(b) numerical values obtained from step (h);

(l) selecting a DBP with an acceptable R_(b) value.

According to the instant invention, R_(b), as defined in (h) above for both the design method and computer system, has a lower limit of 10,000. Preferably R_(b) is greater than 10⁶.

Preferred embodiments of this aspect of the invention are:

1) the computer system as set forth above wherein the DBP R_(b) numerical value is the highest numerical value for all DBP's in step (h) that bind to the target DNA sequence.

2) the computer system above wherein the DBP R_(b) numerical value determined in step (h) is at least 10,000.

3) the computer system above wherein the number of ZF domains, n_(d), is nine.

4) the computer system above wherein the rules for assigning base-contacting amino acids at Z₁, Z₂ and Z₃ for each nucleotide block in step (e) are selected from rule set A.

The method and computer system of the instant invention are further illustrated by the block flow diagrams of FIGS. 4-9.

FIG. 4 shows the components of the computer system on which the DBP design process is implemented. A Central Processor Digital Computer (1) of any manufacture is provided with a Computer Program (2) written by the inventors. This Computer Program (2) reads a series of files described as DNA-Triple Energy Rules (6), Genome Descriptors (9), Genomic DNA Sequence (10) and Gene Features (5). The Central Processor (1) transforms this information into the DBP Blocking Fragment Files (7) and the Optimal DBP Designs for Genome (8).

FIG. 5 shows that the Computer Program (2) in FIG. 4 has two portions. The genomic data is first transformed by the Process Genome into Blocking Fragment Files function (2). These files are then used by the Design DBP's for a Genome function (3).

The Process Genome into Blocking Fragment Files block (2) of FIG. 5 is represented in greater detail in FIG. 6. For every n_(d) from 11 down to 3 the Genome Descriptors file (12) and the Genome DNA Sequence file (32) are read and transformed into the Unsorted Fragment File (7). This same Unsorted Fragment File (14) is transformed by the Sort function (13) provided by the computer manufacturer into the Sorted Fragment file (15). The same Sorted Fragment File (30) is read and transformed eventually into the DBP-Size Blocking File (22).

The Design DBP's for a Genome block (3) of FIG. 5 is represented in greater detail in FIG. 7. The Genome Descriptors file (3), the Gene Features file (7), the Genome DNA Sequence file (9) and the DBP-Size Blocking Files (37) corresponding to the n_(d)'s from 11 down to 3 are read and used to transform the genomic DNA first into genes and then into a file of the Optimal DBP Designs for a Genome (38). The transformation and design process is done for all the genes in a genome.

The “Determine if Current-Sub-Window is in Current-Blocking-File” block (22) in FIG. 7 is expanded in greater detail in FIG. 8.

The “Calculate Binding-Energy-of-Blocking-Fragment” block (24) in FIG. 7 is expanded in greater detail in FIG. 9.

By applying the algorithm to a variety of DBP's of varying n_(d), it was experimentally determined that a value for n_(d) of 9 is the best starting point in the algorithm, i.e., the process should begin with the search for 9-finger DBP's. This can be better understood in terms of the selection criterion, R_(b), used in evaluating various DBP's. In short DBP's, e.g., ones wherein n_(d)=4 or 5, Binding Energy block, which increases geometrically as the product of all Binding Energy_(domain)'s, is significantly lower, and Binding Energy_(site n) values are relatively large. However, as n_(d) increases, the numerator of R_(b) increases dramatically, while, it has been observed, the denominator, representing “background” or “noise,” does not significantly change. Thus, the case of n_(d)=9 provides assurance of high affinity and specificity of binding without also bringing on the possibility of undue computational needs.

However, it should also be emphasized that the present invention is not limited to the design of DBP's wherein n_(d)≦9. For that matter, it will also be appreciated that, while n_(d)=9 has been found to be the best starting point, the best DBP for a given situation may turn out to be one wherein n_(d)<9, the length of the target DNA sequence notwithstanding. The concept of the invention can be applied to the design of DBP's of any length as required.

In any event, for a given DNA sequence of N nucleotides, there are N-27, 9-finger DBP sequences. Each of these can be ordered in terms of strength of binding by evaluating the energy function for each 3-nucleotide segment as set forth in part (e) of the design method disclosed above.

In initial computational experiments, a selectable sequence could have no 8, 7, 6, 5, and 4-finger subsites; however, with the present system, only the sum of the subsite binding energies need be minimized. As a result, it does not matter whether the subsite binding energy comes from 3-finger subsites, 4-finger subsites or even (in principle) larger subsites. This simple change from logical exclusion to energetic exclusion has been mandated not so much by examination of the yeast genome, but more by examination of the worm genome.

The central portion of the instant algorithm is, in the case of finding an acceptable n_(d)-finger site (e.g., a 27-base segment for a 9-finger DBP), the search against all other n_(d)-finger sites in the entire genome to see if there are any similar sites. If such turns out to be the case, the DBP with the highest R_(b) value is selected. Furthermore, the algorithm checks to see if there are any equivalent 8-finger, 7-finger, 6-finger, 5-finger and 4-finger subsites in the whole genome for a given 9-finger site. In the event no acceptable 9-finger site is found, the algorithm then searches for a suitable 8-finger site. If necessary, the search is continued for a 7-finger site and so on, until an acceptable DBP binding site is found.

Within the search for a 9-finger DBP, the algorithm looks at all 27-base sequences, which are called “frames.” Each frame is evaluated to determine its interaction with DNA and the interaction of all other subframes down to 3-finger subsites. The number of instances of each frame and subframe in the genome has been recorded during the genome processing phase of the execution of the software. The sequence of the frame or subframe is evaluated as a product of the binding energy of each ZF. Each ZF domain recognizes three DNA bases. The underlying DNA sequence that a ZF recognizes determines how many hydrogen bonds, water contacts and hydrophobic contact exist between the ZF and the DNA.

The way the algorithm detects whether a given n,-base site occurs in other places in the genome is by looking in a B-tree for the site. The whole genome is processed for each of the n_(d)-finger sites. The algorithm contains means for sorting and merging the myriad fragments and, in the end, there is produced an ordered list of all the blocking fragments for all the different finger sizes.

EXAMPLE 1

The following is given as an example of how the search for, and design of, a DBP is typically carried out. It involves screening for 9-finger DBP's (i.e., n_(d)=9) to bind to a target DNA sequence of 100 nucleotides (i.e, N=100). The sequence is screened, beginning with position 1, for every 27-nucleotide sequence, i.e., 1-27, 2-28, 3-29 etc., in the entire 100-nucleotide sequence. Once this has been done, the 9-fingers are broken down into 3-finger sections, i.e., 1-3, 2-4, 3-5 etc. The algorithm scans and looks for relative strengths of binding. The idea is to maximize the ratio of DBP binding to subsite binding, R_(b), thus eliminating those 9-mers interacting with the greatest numbers of subsites.

The algorithm of the present invention was applied to the genomes of S. cerevisiae and C. elegans as illustrated by the following examples:

EXAMPLE 2

The algorithm has been applied to the screening of the yeast genome. Two chromosomes of yeast, containing 110 and 447 genes, respectively, have been processed. For each gene the algorithm selected the n_(d)-finger sequence with the lowest sum of subsite binding energies. In yeast the number of 3-finger blocking fragments is almost maximal (i.e., 4⁹, versus 4¹² maximal). In the worm genome (see Example 3), the 3-finger blocking sequences are absolutely maximal. In yeast the 4-finger blocking sequences are large in number but the population of 5-finger blocking sequences is relatively small. In worm the 4-finger blocking sequences are larger in number than the 5-finger blocking sequences, but the latter are larger in number relative to yeast. In going in the future from worm to human, one can expect that the 4-finger blocking sequences might come close to saturation (i.e. close to 4¹²) The algorithmic analysis was performed for 2 of the 16 chromosomes of yeast. The 557 cenes in the first two chromosomes seem to present a realistic picture of properties of all the chromosomes in the yeast genome. Sample calculations have been run on the whole yeast genome but these results are not different from those produced by calculating the properties of just two chromosomes' worth of genes. The results of the analysis of 100 yeast genes, typical of the findings throughout the analysis of the yeast genome, are presented in Table 4.

The power of the algorithm is further demonstrated in the results displayed in FIGS. 10-14. The figures display results obtained for all 557 genes of the two yeast chromosomes on which the studies were focused.

The strength of each acceptable 9-finger DBP can be calculated. FIG. 10 shows that the strengths of binding of all the acceptable 9-finger DBP's are uniformly distributed. If this curve were bowed down, then the stronger frames would be more preferred. If this curve were bowed up, then the weaker frames would be preferred.

FIG. 11 shows that the binding energies (Binding Energy_(block)'s) of the acceptable 9-finger DBP's are uniformly distributed between 10¹¹ and 10¹³ binding units.

FIG. 12 shows that the distribution of the sum of the spurious subsite binding energies (Binding Energy_(site)'s) is itself uniform in the range of 10⁶ to 10⁸ binding units.

FIG. 13 is a nonlogarithmic version of FIG. 12. It shows that most of the acceptable 9-finger DBP's have spurious subsite binding energies of less than 5×10⁶.

FIG. 14, produced by taking the ratios of the FIG. 11 values to those of FIG. 12, is a graph of the R_(b)'s for the 9-finger DBP's. This chart shows that the ratio of the DBP binding strength of the acceptable 9-finger DBP's to the sum of the binding energies of the spurious subsite interactions varies from 10⁴ to 10⁶.

The analytical tools of the present invention were also employed in the further analysis of a single yeast gene, YAR073, in particular the 300-bp region of the promoter immediately upstream of the coding region. The full sums of the subsite binding energies (SBE's) for each 27-base frame in this portion of the gene were determined; the results are depicted graphically in FIG. 15. The primary binding energies (BE's) were also determined, and a correlation was found between the SBE values and the values of the ratios of BE:SBE (R_(b)). Still further (FIG. 16), it was seen that the peaks of the plot of the R_(b) values correspond to the footprints of the transcription factors of the same gene (determined in a separate study).

EXAMPLE 3

Application of the algorithm according to the instant invention to 100 genes in C. elegans showed that the system can be applied as successfully to C. elegans as to S. cerevisiae. The results of analysis of the 100 C. elegans genes are presented in Table 5.

In FIG. 17, it can be seen that, for one of the analyzed C. elegans genes, only a 5-finger DBP could be designed. For another gene, only a 7-finger DBP could be designed. These two genes, 2 and 32, are not seen in Table 5, since it presents results of the analysis only for those genes (98 out of 100) for which a 9-mer could be designed. In any event, the results depicted in FIG. 17 are in keeping with the expectation for analysis of the entire C. elegans genome namely, that the distribution of 5- through 9-finger DBP's is somewhat different than in S. cerevisiae.

FIG. 18 represents the same analysis for the C. elegans genes as was depicted in FIG. 14 for S. cerevisiae genes. FIG. 18 shows a similar R_(b) value distribution to that seen in FIG. 14.

Examples 2 and 3 demonstrate the applicability of the instant invention to the design of DBP's for the genomes of two widely disparate organisms. The various results of the application of the algorithm to the yeast genome, in particular, and also to the worm genome, show the power of the algorithmic tool and demonstrate its foundation in reality, i.e., that it does not merely provide a random and/or theoretical analysis. It is to be expected, on the basis of these analyses, that the inventive algorithm can be extended to the design of DBP's for any desired segment of the genome of any organism of interest, including that of a human.

Although the instant algorithm involves a search against the entire genome of an organism, the results of the present studies strongly indicate that lack of complete knowledge of the genome of a given organism would not constitute an impediment to application of the present invention to the design of DBP's for that organism. One would expect to be able to use the knowledge of block sequences obtained in the studies presented herein on S. cerevisiae (a unicellular organism) and C. elegans (a multicellular organism) to form valid estimates of allowable sequences for the systems of higher eukaryotes.

For example, the present studies on yeast and worm indicate that the genomic “noise,” in this context the spurious binding site energies, is relatively constant, and this can be projected to higher, more complex organisms as well. In other words, one would expect from the demonstrated combinatorics of DNA sequences to be able to extrapolate, or extend, the present algorithm to the analysis of more complex genomes, however much is known of the specific sequences therein, with the object of designing effective DBP's. Furthermore, as the entire genomes of larger organisms, e.g., D. melanogaster, become known, they will provide further keys to the analysis of the genomes of higher organisms, including humans.

A DBP as specified above may be built by using standard protein synthesis techniques; or, employing the standard genetic code, may be used as the basis for specifying and constructing a gene whose expression is the DBP.

Proteins so designed can be used in any application requiring accurate and tight binding to a DNA target sequence. For example, a DBP, according to the instant invention, can be coupled with a DNA endonuclease activity. When the resultant molecule binds to the target DNA, said DNA can be cut at a fixed displacement from the DBP binding site.

Similarly, in instances in which the target DNA sequence is a promoter, one can produce a promoter-specific DBP which, when bound, will act to alter (i.e., enhance, attenuate or even terminate) expression of a given gene or, alternatively, genes under control of that promoter.

As another application, a DBP could be designed to bind specific DNA sequences when attached to solid supports. Such solid supports could include styrene beads, acrylamide well-plates or glass substrates.

In order to realize the specific applications mentioned above, as well as the full scope of applications possible through the instant invention, the DBP can be designed as set forth above to include the added feature of a pre- and/or postdomain amino acid sequence of arbitrary length. This would include, for example, the coupling of the basic DBP to an endonuclease or to a reporter or to a sequence by which the DPB could be coupled to a solid support.

Accordingly, the instant invention includes DBP's that bind to a predetermined target double-stranded DNA sequence of 3n (where n≧1) base pairs in length of the form:

NH₂—X_(0-m)—ZiF₁—[{linker}—ZiFi] . . . —[{linker}—ZiF_(n)]-X_(0-p)—COOH

wherein each ZiF₁ to ZiF_(n) is a ZF domain of the form set forth above; {linker} is an amino acid sequence as set forth above; X_(0-m) stands for a sequence of from 0 to m amino acids and X_(0-p) stands for a sequence of from 0 to p amino acids. The values for m and p and the identities of the amino acids are determined by the particular protein(s) or amino acid sequence(s) to be coupled to the DBP for a given application.

In a further embodiment of the invention, the Zn⁺² atom, which forms a complex with the two cysteine and two histidine amino acids in a specific ZF motif, can be substituted by a Co⁺² or a Cd⁺² atom, thus making a “cobalt finger” or a “cadmium finger.”

The rules presented in Table 2 (“rule set A”) are to be regarded as the “first choice” rules for optimal combinations in ZF-DNA recognition. However, it should be emphasized, as indicated in column B (“rule set B”) of Table 1 or Table 3, that there are many alternative AA combinations that would also be expected to be important in the design of DNA-binding proteins capable of forming useful ZF-DNA complexes.

TABLE 4 Scanning the complete S. Cerevisiae genome for conflicts - DBP's designed against the coding region of each gene Ratio of Binding Energy to Specific Amino Acids in DBP DBP Spurious Chrom- Zf1 Zf2 Zf3 Zf4 Zf5 Zf6 Zf7 Zf8 Zf9 Binding Binding osome Gene Optimal DNA Sequence in Gene* Z1 Z2 Z3 Z1 Z2 Z3 Z1 Z2 Z3 Z1 Z2 Z3 Z1 Z2 Z3 Z1 Z2 Z3 Z1 Z2 Z3 Z1 Z2 Z3 Z1 Z2 Z3 Energy Energy 1 1 CCT ACT CTC AGA TTC CAC TTC ACT CCA E N3 Q2 Q N3 Q2 E I R2 Q N Q I I R2 E N E I I R2 Q N3 Q2 E E Q  7374132  132836 1 2 TCA GAG CTC ACT TAG CCC AAT ACT ACA I D Q R N R E I R2 Q N3 Q2 I N R E Q3 R2 Q N Q3 Q N3 Q2 Q D Q 24192270  166073 1 3 GCC GCT TGG GAC GTC GGA GAA GGA GCC R Q3 R2 R N3 Q2 I H R R N Q3 R I R2 R D Q R N Q R N Q R Q3 R2  8480520  701860 1 4 GAA AAG CAT ACA GCT TDC GAT ACA TCA R N Q Q Q R E Q1 02 Q D Q R N3 Q2 I Q3 R2 R Q Q2 Q D Q I D Q 19226970  313442 1 5 AGT AGC ATA ATA GGA TCT AGT ACT GDC Q H1 Q2 Q H1 R2 Q I Q Q I Q R N Q I1 N3 Q2 Q H1 Q2 Q N3 Q2 R Q3 R2 15168192  176738 1 6 GCC GTG GAA ACA ATT GCA GAG GAG ATG R Q3 R2 R I R R N Q Q D Q Q I Q3 R D Q R N R R N R Q I R 30941379 176738 1 7 CCA AGT CGA GAC GCT TAA AGT TAA GTG E D Q Q H1 Q2 E N Q R N Q3 R N3 Q2 I N Q Q H1 Q2 I N Q R I R  9355725  320720 1 8 GAC TAC ACC GGC ATG CCT GDC AGC AAA R N Q3 I N E Q H1 R2 R H1 R2 Q I R E N3 Q2 R Q3 R2 Q H1 R2 R N Q  6539130  487737 1 9 ACT TGG ACT GCC GAG CGG GTC GAT CAA Q N3 Q2 I H R Q N3 Q2 R Q3 R2 R N R E H R R I R2 R Q Q2 E N Q3  442890 1094811 1 10 GAC CCA ACT GCG CAC GCT GDC AAG GAG R N Q3 E D Q Q N3 Q2 R E R E N E R N3 Q2 R Q3 R2 Q Q R R N R 11213640 1011037 1 11 CCT GCC AGC AGT TGT CCG GAC CTG CAT R N3 Q2 R Q3 R2 Q H1 R2 Q H1 Q2 I H1 Q2 R E R R N Q3 E I R E Q1 Q2  6292670  563027 1 12 GAT AAC ACA GTG CAG AAG ACT CCT ACA R Q Q2 Q Q1 R2 Q D Q R I R E Q R Q Q R Q N3 Q2 E N3 Q2 Q D Q 18371043  492068 1 13 TAT GCA DCC GAT ACA GAG GGT GTC GAG I N Q3 R D Q E Q3 R2 R Q Q2 Q D Q R N R R H Q2 R I R2 R N R  7619823  768933 1 14 CAA GAG CAC GAC GGC CGC AGT AAG CAC E N Q3 R N R E N E R N Q3 R H1 R2 E H1 R2 Q H1 Q2 Q Q R E N E  4512410 1279511 1 15 GAG ATA CCC GTG ATA CGT CGC GGT AAA R N R Q I Q E Q3 R2 R I R Q I Q E H Q2 E H1 R2 R H Q2 R N Q  2512107  903296 1 16 AAT GGG GCA CGA CCT DCA GAG GGT GAT Q N Q3 R H R R D Q E N Q E N3 Q2 E D Q R N R R H Q2 R Q Q2 10115586  700491 1 17 GAG AGA CDC CAG GCG TGA GAC ACG TCT R N R Q N Q E Q3 R2 E Q R R E R I N Q R N Q3 Q E R I1 N3 Q2  4430376 1101825 1 18 ACC TCC GCT GTC ATG GGT ACG GGT GGC Q Q3 R2 I Q3 R2 R N3 Q2 R I R2 Q I R R H Q2 Q E R R H Q2 R H1 R2  4927770  713565 1 19 GAA CCG AGT TAG GGG CGA TCT AAG CGA R N Q E D R Q H1 Q2 I N R R H R E N Q I1 N3 Q2 Q Q R E N Q  2905470 1180262 1 20 GAT GGA ACA CCC AAG GGT CCT CGT GAA R Q Q2 R N Q Q D Q E Q3 R2 Q Q R R H Q2 E H Q2 E H Q2 R N Q 11022512  583391 1 21 ACC GAC CAT ACA GGA CGT CCT GDC ACC Q Q3 R2 R N Q3 E Q1 Q2 Q D Q R N Q E H Q2 R N3 Q2 R Q3 R2 Q Q3 R2  9693576 1085516 1 22 GAA AGA CGG AAG CTC GAT CCT AAC GCT R N Q Q N Q E H R Q Q R E I R2 R Q Q2 R N3 Q2 Q Q1 R2 R N3 Q2 12066840  645941 1 23 AGT CAA CTG GGA AGG GTC CGG CAT CAT Q H1 Q2 E N Q3 E I R R N Q Q Q R R I R2 E H R E Q1 Q2 E Q1 Q2  7060608  422504 1 24 AGG ATG GTA CGT GGC ACG GGA CCA TCT Q Q R Q I R R I Q E H Q2 R Q3 R2 Q E R R N Q E D Q I1 N3 Q2  4607532  626666 1 25 ATG GAC CAC CCG CAT GCC CGC TGT GAG Q I R R N Q3 E N E E D R E Q1 Q2 R Q3 R2 E H1 R2 I H1 Q2 R N R  2450880 1266455 1 26 AAG ACC CDC ACG CCC GTG TCC GCA CCT Q Q R Q Q3 R2 E Q3 R2 Q E R E N3 Q2 R I R I Q3 R2 R D Q E N3 Q2  5803812  981266 1 27 GAT CAG CCC GCC GGC CCT GGT GCG GAC R Q Q2 E Q R E Q3 R2 R Q3 R2 R H1 R2 E N3 Q2 R H Q2 R E R R N Q3  2742660 3269843 1 28 AGT CCG CAC GAG GGC CGG CGG GCT GAT Q H1 Q2 E D R E N E E Q R R H1 R2 E H R E H R R N3 Q2 R Q Q2  2263544 1870532 1 29 AAT GGC GCT AAC CGG GAC AGT AGC GAC Q N Q3 R H1 R2 R N3 Q2 Q Q1 R2 E H R R N Q3 Q H1 Q2 Q H1 R2 R N Q3  6611550 1263414 1 30 GCC ACT GCC GDC TCT GTC AGC GCT GCC R Q3 R2 Q N3 Q2 R Q3 R2 R Q3 R2 I1 N3 Q2 R I R2 Q H1 R2 R N3 Q2 R Q3 R2 10878345  738828 1 31 ACT CCG AGG CGA GDG CAA AGC GGA ACA Q N3 Q2 E D R Q Q R E N Q R E R E N q3 Q H1 R2 R N Q Q D Q  4999968 1239753 1 32 ACC GTC GCT GCC TCC GCT GTC GCT GCT Q Q3 R2 R I R2 R N3 Q2 R Q3 R2 I Q3 R2 R N3 Q2 R I R2 R N3 Q2 R N3 Q2   9376290  720036 1 33 ACG TAG CCA GAC GAG CTC AGT CAA GAG Q E R I N R R D Q R N Q3 R N R E I R2 Q H1 Q2 E N Q3 R N R  6475788  77552 1 34 ACC ACC CTG GTT ACC GTC DCC GGT GTC Q Q3 R2 Q Q3 R2 E I R R I Q3 Q Q3 R2 R I R2 E Q3 R2 R H Q2 R I R2  3659040  830292 1 35 ACA ACC CAG GAA AAC GCC TCC GAA GCC Q D Q Q Q3 R2 E Q R R N Q Q Q1 R2 R Q3 R2 I Q3 R2 R N Q R Q3 R2 15634620  713817 1 36 CAA CAA GCG AGA TGG GCG GAT ATC CCA E N Q3 E N Q3 R E R Q N Q I H R R E R R Q Q2 Q I R2 E D Q  6125355  566708 1 37 GCC GGA TGC GAG GAC GCA AGC AGG GGA R Q3 R2 R N Q I1 H1 R2 R N R R N Q3 R D Q Q H1 R2 Q Q R R N Q  5445990 1053906 1 38 AAA GGT ACC GCT ACG CCA CCT ACG GGT R N Q R H Q2 Q Q3 R2 R N3 Q2 Q E R E D Q E N3 Q2 Q E R R H Q2  6989373  950445 1 39 ACT GAT CGT GAA CCC CGT CAA GGT AAG Q N3 Q2 R Q Q2 E H Q2 R N Q E Q3 R2 E H Q2 E N Q3 R H Q2 Q Q R  9882648  722977 1 40 CAA CGG GAC TCC GCA GAC GGG AGC AAT E N Q3 E H R R N Q3 I Q3 R2 R D Q R N Q3 R H R Q H1 R2 Q N Q3  3614610 1572359 1 41 CCC GAG GAG GTA CCC CTA GAT CAC TAT E Q3 R2 R N R R N R R I Q E Q3 R2 E I Q R Q Q2 E N E I N Q3  5069952  739622 1 42 GAC CCT TAT GCT CTA TCC GAG CAC GAT R N Q3 E N3 Q2 I N Q3 R N3 Q2 E I Q I Q3 R2 R N R E N E R Q Q2  6148980  677592 1 43 CAA GGT GGA CAG CCG AAC ATA GCT GGT E N Q3 R H Q2 R N Q E Q R E D R Q Q1 R2 Q I Q R N3 Q2 R H Q2  6691509  833912 1 44 CCG GAT TAC ACG TCT GCC TCG ACC GCA E D R R Q Q2 I N E Q E R I1 N3 Q2 R Q3 R2 I D R Q Q3 R2 R D Q 27652514 1034213 1 45 GGT GCC GAT ACG GAT AAT GCG GTA ACT R H Q2 R Q3 R2 R Q Q2 Q E R R Q Q2 Q N Q3 R E R R I Q Q N3 Q2 11200230  830360 1 46 ATC AGC GAC TCT AGG CCG CAC GTT CAG Q I R2 Q H1 R2 R N Q3 I1 N3 Q2 Q Q R E D R E N E R I Q3 E Q R  3766644  774309 1 47 ACG CCT GAA AGA GCG CAC ACT CCT GCC Q E R E N3 Q2 R N Q Q N Q R E R E N E Q N3 Q2 E N3 Q2 R Q3 R2 10318266  901129 1 48 TCT AGT GCC CGG AAC ACA CQG AGA GCA I1 N3 Q2 Q H1 Q2 R Q3 R2 E H R Q Q1 R2 Q D Q E H R Q N Q R D Q  3231252 1293053 1 49 AGC GCT GAT GAG AGA GAC GCG GAA GAT Q H1 R2 R N3 Q2 R Q Q2 R N R Q N Q R N Q3 R E R R N Q R Q Q2 17979930  777874 1 50 ACC GCC GCA CCA ACG GCA CTC GCC ACG Q Q3 R2 R Q3 R2 R D Q E D Q Q E R R D Q E I R2 R E R Q E R  5487912 1016572 1 51 ACG GGA GAT AGC ACT CCC TCA GGC ACG Q E R R N Q R Q Q2 Q H1 R2 Q N3 Q2 E Q3 R2 I D Q R H1 R2 Q E R  5807160  9266587 1 52 GAC GAG GGC GGC CGC ATA GTG CAC GCA R N Q3 R N R R H1 R2 R H1 R2 E H1 R2 Q I Q R I R E N E R D Q  2579344 1371038 1 53 ACC GCT GGC GCA GAC GCC ACT ACC AAG Q Q3 R2 R N3 Q2 R H1 R2 R D Q R N Q3 R Q3 R2 Q N3 Q2 Q H1 R2 Q Q R  7686450 1600463 1 54 TAT GAG CCG TAC CAG ATA CGT GCT AAT I N Q3 R N R E D R I N E E Q R Q I Q E H Q2 R N3 Q2 Q N Q3  6770394  401149 1 55 CAG ACA CCA CCG AGC CCC GAT CAA GAG E Q R Q D Q E D Q E D R Q H1 R2 E Q3 R2 R Q Q2 E N Q3 R N R  9717264  708788 1 56 CAT CGC GTT GGC ACT CGG TCC CGA AAG E Q1 Q2 E H1 R2 R I Q3 R H1 R2 Q N3 Q2 E H R I Q3 R2 E N Q Q Q R  2937552 1059390 1 57 GCT ATT GGG CCT GCC CGG TGT ACG GCC R N3 Q2 Q I Q3 R H R E N3 Q2 R Q3 R2 E H R I H1 Q2 Q Q R R Q3 R2  3094125 1175589 1 58 ATT CCT GAT ACG GCG GTT GAC AAG GAG Q I Q3 E N3 Q2 R Q Q2 Q E R R E R R I Q3 R N Q3 Q Q R R N R 12276765  572705 1 59 GAC GCC AGG GAC GAG CAA GGG GAC GAA R N Q3 R Q3 R2 Q Q R R N Q3 R N R E N Q3 R H R R N Q3 R N Q  6852600 1938830 1 60 GTC GCC AAC GCT CAC GGT GTG GAA ACC R I R2 R Q3 R2 Q Q1 R2 R N3 Q2 E N E R H Q2 R I R R N Q Q Q3 R2  7299000  880912 1 61 AGA AGG GGC AAG CGT GGT CGT GAC GAT Q N Q Q Q R R H1 R2 Q Q R E H Q2 R H Q2 E H Q2 R N Q3 R Q Q2  5511175 1080370 1 62 CAG CAG ACC GAC TAA ACT ACT GCA CGA E Q R E Q R Q H1 R2 R N Q3 I N Q Q N3 Q2 Q N3 Q2 R D Q E N Q 10230030  654559 1 63 CAG CAG ACC GAC TAA ACT ACT GCA CGA E Q R E Q R Q H1 R2 R N Q3 I N Q Q N3 Q2 Q N3 Q2 R D Q E N Q 10230030  654559 1 64 GAT GTG GGG AAC CAT GCC CAG GAT GAT R Q Q2 R I R R H R Q Q1 R2 E Q1 Q2 R Q3 R2 E Q R R Q Q2 R Q Q2 10264770  906033 1 65 ACT GTG GGC AAC AGT ACG GCA ATT ACC Q N3 Q2 R I R R H1 R2 Q Q1 R2 Q H1 Q2 Q E R R D Q Q I Q3 Q Q3 R2 10593486  512119 1 66 ACT GAC GAG CAT GAA GCT GAT GTC AAT Q N3 Q2 R N Q3 R N R E Q1 Q2 R N Q R N3 Q2 R Q Q2 R I R2 Q N Q3 31895100  3888784 1 67 GAC CCA GAA GTG CAG GCT GAC ATG AAG R N Q3 E D Q R N Q R I R E Q R R N3 Q2 R N Q3 Q I R Q Q R 14004360  495857 1 68 GAT CAT TAG CAA AGT GAG CAA CAC ACG R Q Q2 E Q1 Q2 I N R E N N3 Q H1 Q2 R N R E N Q3 E N E Q E R 21159954 2844809 1 69 GAT CGG AAG TAA GAC CCC CGA TAT GAC R Q Q2 R H R Q Q R I N Q R N Q3 E Q3 R2 E N Q I N Q3 R N R  8164692  708759 1 70 GGT GCT CGG CAT ACA GCC CTG ATT GTT R H Q2 R N3 Q2 E H R E Q1 Q2 Q D Q R Q3 R2 E I R Q I Q3 R I Q3  4056912  706071 1 71 CAT GTC AGC ACT GCA CAG CAG CCG AAA E Q1 Q2 R I R2 Q H1 R2 Q N3 Q2 R D Q E Q R R N R E D R R N Q  5930865  940863 1 72 GCT ACC GCT ACT GTG CCT TCC GCT CCC R N3 Q2 Q H1 R2 R N3 Q2 Q N3 Q2 R I R E N3 Q2 I Q3 R2 R N3 Q2 E Q3 R2 10216521  637218 1 73 GAT CGT GAA GCT ACC TTT AGA GAC GCC R Q Q2 E H Q2 R N Q R N3 Q2 Q Q3 R2 I I Q3 Q N Q R N Q3 R Q3 R2 11804220  440208 1 74 AAC GTT GCT GAG AGT CCT GGA ACG GGC Q Q1 R2 R I Q3 R H Q2 R N R Q H1 Q2 E N3 Q2 R N Q Q Q R R H1 R2 11792148  492924 1 75 GCT GAA GCC GAC TAT CTT GCC GAT GAG R N3 Q2 R N Q R Q3 R2 R N Q3 I N N3 E I Q2 R Q3 R2 R Q Q2 R N R  9898740  835143 1 76 GAA TAA CAT TAG ACC GGG GCG CCA AAC R N Q I N Q E Q1 Q2 I N R Q H1 R2 R H R R E R E D Q Q Q1 R2  9295611  466898 1 77 CAA TGC ACA GAC AAA TTC GAT ACT CAC E N Q3 I1 H1 R2 Q D Q R N Q3 R N Q I I R2 R Q Q2 Q N3 Q2 E N E 38166822   59410 1 78 No DBP possible 1 79 GCT AAT CAC GTC GAC GTC GGG CAT GGA R N3 02 Q N Q3 E N E R I R2 R N Q3 R I R2 R H R E Q1 Q2 R N Q  9510655  945100 1 80 CCT GCC CAG GCC GCT GAG CTG GTG GGT E N3 Q2 R Q3 R2 E Q R R Q3 R2 R N3 Q2 R N R E I R R I R R H Q2  5592240  997835 1 81 ACC CAG GCA GTG CAG GAG CGA GCC AGG Q Q3 R2 E Q R R D Q R I R R N R R N R E N Q R Q3 R2 Q Q R  6631281  1211740 1 82 CCA GGG GCT CCC GCT CCC TGG GCA GAA E N Q R E R R N3 Q2 E Q3 R2 R N3 Q2 E Q3 R2 I H R R D Q R N Q  4854492  1135027 1 83 GTG CCT CCG CCG CAG CAA CCA CTA CAT R I R E N3 Q2 E D R E D R E Q R E N Q3 E D Q E I Q E Q1 Q2 10134072  274029 1 84 CCT GAT GTA CAG TGG GAT ACA GCG AAT E N3 Q2 R Q Q2 R I Q E Q R I H R R Q Q2 Q D Q R E R Q N Q3 10593990  455091 1 85 CCA ACG AAC GGT GCA CAG AAT ACG CAG E D Q Q E R Q Ql R2 R H Q2 R D Q R N R Q N Q3 Q E R E Q R 16355520  526388 1 86 GGT GAT CGC ACG CAA GCC TGC GGG GAA R H Q2 R Q Q2 E H1 R2 Q E R E N Q3 R Q3 R2 I1 H1 R2 R H R R N Q  2939586 1473085 1 87 GAG AAG CCA ACA AGC AAC GCT GAG GAA R N R Q Q R E D Q Q D Q Q H1 R2 Q Q1 R2 R N1 Q2 R N R R N Q 20535210  575100 1 88 GAG GCG ACA TAA GTT GTT AGC ACA GCA R N R R E R Q D Q I N Q R I Q3 R I Q3 Q H1 R2 Q D Q R D Q 14004180  263582 1 89 GAT CAT GGG CTA TGG GAT ACT CCC TAC R Q Q2 E Q1 Q2 R H R E I Q I H R R Q Q2 Q N3 Q2 E Q3 R2 I N E   5240610  432675 1 90 AAG TTA GAT ACA CTT AAC GAA GCT AGT Q Q R I I Q R Q Q2 Q D Q E I Q2 Q Q1 R2 R N Q E N3 Q2 Q H1 Q2 17980110  177343 1 91 AAT GCT CCC AGG GGC TCG GCC AGG ACC Q N Q3 R N3 Q2 E Q3 R2 Q Q R R H1 R2 I D R R Q3 R2 Q Q R Q Q3 R2  4905765 1462454 1 92 GAT TCG CCA CGG CAC ACT CCG CAC AGA R Q Q2 I D R E D Q E H R E N E Q N3 Q2 E D R E N E Q N Q  2361600  1351886 1 93 GCG GAG TGA GCC GCC CAG GCC AAC GAC R E R R N R I N Q R Q3 R2 R Q3 R2 R N R R Q3 R2 Q Q1 R2 R N Q3 9558540 1111971 1 94 ACA TCG CCG CCT ACC AGT CCC GCG GTG Q D Q I D R E D R E N3 Q2 Q Q3 R2 Q H1 Q2 E Q3 R2 R E R R I R  3132513 1154321 1 95 GGT ATC GCC AGT CTC CGG GTC CAT ACC R H Q2 Q I R2 R Q3 R2 Q H1 Q2 E I R2 E H R R I R2 E Q Q2 Q Q3 Q2  4725558  561264 1 96 ATC ACT CCC GGT CCA CAT TCT AGG AAG Q I R2 Q N3 Q2 E Q3 R2 R H Q2 E D Q E Q1 Q2 I1 N3 Q2 Q Q R Q Q R  6608547  709280 1 97 AAT GTC AGT TGT GAC GCT ACA CTT TTC Q N Q3 R I R2 Q H1 Q2 I H1 Q2 R N Q3 R N3 Q2 Q N Q E I Q2 I I R2  8150676  162281 1 98 AAT ACT GTA GCT GCT GAG ACG ATT ACC Q N Q3 Q N3 Q2 R I Q R N3 Q2 R N3 Q2 R N R Q E R Q I Q3 Q Q3 R2 18607320  466494 1 99 AGC TGT GGA AAC AAT CGC AGG GGA GAT Q H1 R2 I H1 Q2 R N Q Q Q1 R2 Q N Q3 E H1 R2 Q Q R R N Q R Q Q2 19293534  242396 1 100 GTA GAA ATC TGC TGC ACA TGC CAC ACG R I Q R N Q Q I R2 I1 H1 R2 I1 H1 R2 Q D Q I1 H1 R2 E N E Q E R  9178908  103292 1 104 GCT ACG GAG AAA CCG TGC ACT AAC TCT R N3 Q2 Q E R R N R R N Q E D R I1 H1 R2 Q N3 Q2 Q Q1 R2 I1 N3 Q2 10532100  418624 1 105 ACC GTT TCC TCC AAG TCA TAC ACC ACT Q Q3 R2 I Q3 R2 I Q3 R2 I Q3 R2 Q Q R I D Q I N E Q Q3 R2 Q N3 Q2 15664500  137917 1 107 CTA TAT ATT TGT GGC AGA AAT CAT ACC E I Q I N Q3 Q I Q3 I H1 Q2 R H1 R2 Q N Q Q N Q3 E Q1 Q2 Q Q3 R2 36455616   57145 1 108 TAT CAT GAC AAC ATG GTA CAA ATT GAG I N Q3 E Q1 Q2 R N Q3 Q Q1 R2 Q I R R Q E N Q3 Q I Q3 R N R 25006833  153077 1 109 AAG ACC GCA CTA GAT CTT ACC AAG AGC Q Q R Q Q3 R2 R D Q E I Q R Q Q2 E I Q2 Q Q3 R2 Q Q R Q H1 R2 13895100  382466 1 110 ATG ACG ACA TCC AAG CCA GCT TTT ACC Q I R Q E R Q D Q I Q3 R2 Q Q R E D Q R N3 Q2 I I Q3 Q Q3 R2 16928244  147676 *The optimal DNA sequences in genes 1-61 are shown as SEQ ID NOS: 5-65, respectively, in the Sequence Listing. The sequences in genes 62 and 63 are identica1 and shown as SEQ ID NO: 66. The sequences in genes 64-77 are shown as SEQ ID NOS: 67-80, respectively. The sequences in genes 79-100 are shown as SEQ ID NOS: 81-102, respectively. The sequences in genes 104 and 105 are shown as SEQ ID NOS: 103 and 104, respectively. The sequences in qenes # 107-110 are shown as SEQ ID NOS: 105-108 respectively.

TABLE 5 Scanning the complete C. elegans for conflicts - DBP's designed against the coding region of each gene Ratio of Binding Energy to Specific Amino Acids in DBP DBP Spurious Chrom- Zf1 Zf2 Zf3 Zf4 Zf5 Zf6 Zf7 Zf8 Zf9 Binding Binding osome Gene Optimal DNA Sequence in Gene* Z1 Z2 Z3 Z1 Z2 Z3 Z1 Z2 Z3 Z1 Z2 Z3 Z1 Z2 Z3 Z1 Z2 Z3 Z1 Z2 Z3 Z1 Z2 Z3 Z1 Z2 Z3 Energy Energy 1 1 ACC AAG CCG CCC GAC TCC TAG GAG ATG Q E R Q Q R E D R E Q3 R2 R N Q3 I Q3 R2 I N R R N R Q I R 23846400 183458 1 3 GAG GCC GAG CAC GAG CAC AGG AGC GAA R N Q3 R Q3 R2 R N R E N E R N R E N E Q Q R Q H1 R2 R N Q 58257900  162173 1 4 GAC AAT TCT CCC GGC GCC AAT GAT CAC R N Q3 Q N Q3 I1 N3 Q2 E Q3 R2 R H1 R2 R Q3 R2 Q N Q3 R Q Q2 E N E 83021040  99575 1 5 AGC ACG ACT CGG CAC ACC CGT TCC GCC Q H1 R2 Q E R Q N3 Q2 E H R E N E Q Q3 R2 E H Q2 I Q3 R2 R Q3 R2 25095960 177149 1 6 CCA GGA AGG CGC ACT GGC AGA CGT AAG E D Q R N Q Q Q R E H1 R2 Q N3 Q2 R H1 R2 Q N Q E H Q2 Q Q R 34627332 148563 1 7 CCT ATT ACT CGT GGT GCC GCG GGA GCC R N3 Q2 Q I Q3 Q N3 Q2 E H Q2 R H Q2 R Q3 R2 R E R R N Q R Q3 R2 32769390 208126 1 8 AAT AGC GAA CAG ATA TCT GAC AAC TCC Q N Q3 Q H1 R2 R N Q E Q R Q I Q I1 N3 Q2 R N Q3 Q Q1 R2 I Q3 R2 85891770  58952 1 9 GTC GCG CAT ACT AAG GCT CGC TTT AAT R I R2 R E R E Q1 Q2 Q N3 Q2 Q Q R R N3 Q2 E H1 R2 I I Q3 Q N Q3 22607199 144807 1 10 GAT ATT CGC GAT GGC AGC GGT GAC GAT R Q Q2 Q I Q3 E H1 R2 R Q Q2 R H1 R2 Q Q3 R2 R H Q2 R N Q3 R Q Q2 35024550 200334 1 11 CAT AAC GTC GAG GCT GCC CGC AAG GAG E Q1 Q2 Q Q1 R2 R I R2 R N R R N3 Q2 R Q3 R2 E H1 R2 Q Q R R N R 26896050 338100 1 12 ACC AGC CAT CAC GCC ATG CGA AGC ACC Q Q3 R2 Q H1 R2 E Q1 Q2 E N E R Q3 R2 Q I R E N Q Q H1 R2 Q Q3 R2 35287740 156180 1 13 AGG CAC CCC GGG AAG CGC CGG ACC GAA Q Q R E N E E Q3 R2 R H R Q Q R E H1 R2 E H R Q Q3 R2 R N Q 14482326 394684 1 14 GGA GCA CCA GAC GCC CCG ACT AAG CCG R N Q R D Q E D Q R N Q3 R Q3 R2 E D R Q N3 Q2 Q Q R E D R 37857024 21565 1 15 GAT GCT CGC CTA CTC GCG AGG CCA AGA R Q Q2 R N3 Q2 E H1 R2 E I Q E I R2 R E R Q Q R E D Q Q N Q 20671398 146603 1 16 AGA GCC GAT GCC CGC ACC AAG GCT GCT Q N Q R Q3 R2 R Q Q2 R Q3 R2 E H1 R2 Q Q3 R2 Q Q R R N3 Q2 R N3 Q2 44071200 294768 1 17 ACA GAC GAG GCC AAG ATC TAC GCC GAA Q D Q R N Q3 R N R R Q3 R2 Q Q R Q I R2 I N E R Q3 R2 R N Q 4248410 155670 1 18 CCA ACC GGT AGG CCG ATA GGT ACT CGC E D Q Q Q3 R2 R H Q2 Q Q R E D R Q I Q R H Q2 Q H1 Q2 E H1 R2 21033604 160124 1 19 CCT ACA AGG CCC CGT AGG AGG GCC GAT E N3 Q2 Q D Q Q Q R E Q3 R2 E H Q2 Q E R Q Q R R Q3 R2 R Q Q2 15698286 488647 1 20 ACC AAC GGA CAG GAT GGC GCC GAA CAA Q Q3 R2 Q Q1 R2 R N Q E Q R R Q Q2 R H1 R2 R Q3 R2 R N Q E N Q3 60312960 198256 1 21 GCA AGC CGC TAT GTC GAC GCT CGC CAA R D Q Q H1 R2 E H1 R2 I N Q3 R I R2 R N Q3 R N3 Q2 E H1 R2 E N Q3 22049052 158783 1 22 AGT CCG CCT AAG CGC CCT GTT CCG CCG Q H1 Q2 E D R E N3 Q2 Q Q R E H1 R2 E N3 Q2 R I Q3 E D R E D R 13743972 254069 1 23 AAG CCG ATC GCT GCG ACC GAA CCG CCT Q Q R E H R Q I R2 R N3 Q2 R E R Q Q3 R2 R N Q E D R E N3 Q2 25602930 255727 1 24 AAT GAT CTA GCT CCC AGT CCT ACT GCG Q N Q3 R Q Q2 E I Q R N3 Q2 E Q3 R2 Q H1 Q2 E N3 Q2 Q N3 Q2 R E R 39720375 180624 1 25 CAG AGG ACT CGC CGT ATC GTC GCT GGT E Q R Q Q R Q N3 Q2 E H1 R2 E H Q2 Q I R2 R I R2 R N3 Q2 R H Q2 20131686 173906 1 26 GCT ATG AGG TCA CGG GCT CCC CCT GAT R N3 Q2 Q I R Q Q R I D Q E H R R N3 Q2 E Q3 R2 E N3 Q2 R Q Q2 2813218 193724 1 27 GAC ACA GGT CCC CAT CAC GGG ACA AGT R N Q3 Q D Q R H Q2 E Q3 R2 E Q1 Q2 E N E R H R Q D Q Q H1 Q2 31613868 189117 1 28 CAC GTT GGG ACC GCC AGG AGC CCG AAT E N E R I Q3 R H R Q Q3 R2 R Q3 R2 Q Q R Q H1 R2 E D R Q N Q3 30041550 183454 1 29 CGA GAA CGT CCC ATC AAG CGT GAA CAC E N Q R N Q E H Q2 E Q3 R2 Q I R2 Q Q R E H Q2 R N Q E N E 39080106 109209 1 30 GAT CCT TCG AGT ACG CCT ACG CAA GGA R Q Q2 E N3 Q2 I D R Q H1 Q2 Q E R E N3 Q2 Q E R E N Q3 R N Q 35934354 145548 1 31 AAC GCC GCC TAG ACC CCG GGG AAT ACC Q Q1 R2 R Q3 R2 R Q3 R2 I N R Q Q3 R2 E D R R H R Q N Q3 Q Q3 R2 24227280 379135 1 33 GTG GTG CAG GAG GGG CAG GAG GCC ACA R I R R I R E Q R R N R R H R E Q R R N R R Q3 R2 Q D Q 35599014 164587 1 34 CGT ACA AGG AAT ACC CTG AAG GAC AAC E H Q2 Q D Q Q Q R Q N Q3 Q Q3 R2 E I R Q Q R R N Q3 Q Q1 R2 98170218 71458 1 35 ACC TCC ACT CAG CGA CCA CCG GCG CCA Q Q3 R2 I Q3 R2 Q N3 Q2 E Q R E N Q E D Q E D R R E R E D Q 36901980 123875 1 36 AAA GCG GTA CAA GCG ACG ACC CGT GCT R N Q R E R R I Q E N Q3 R E R Q E R Q Q3 R2 E H Q2 R N3 Q2 24232698 227970 1 37 ACA ACG ACC GAG GAG CCT ACC GAA GCC Q D Q Q E R Q Q3 R2 R N R R N R E N3 Q2 Q Q3 R2 R N Q R Q3 R2 49723200 288576 1 38 GAG AAC CGT CCC CAC CCG AAG CTG GCT R N R Q Q1 R2 E H Q2 E Q3 R2 E N E E D R Q Q R E I R R N3 Q2 25381692 196538 1 39 CGT CCG GTG CCC GAG ACT GGG ACC GGT E H Q2 E H R R I R E Q3 R2 R N R Q N3 Q2 R H R Q Q3 R2 R H Q2 22963398 201668 1 40 AGG GAC CGT ACG GAA CAG GAC CCC CCT Q Q R R N Q3 E H Q2 Q E R R N Q E Q R R N Q3 E Q3 R2 E N3 Q2 29852550 285512 1 41 AGG CAA CCC TGG AGG CGT CCC AGC ACC Q Q R E N Q3 E Q3 R2 I H R Q Q R E H Q2 E Q3 R2 Q H1 R2 Q Q3 R2 16371369 231386 1 42 CTC ACT ACC CCG GCA CCA AGG ACC AGT E I R2 Q N3 Q2 Q Q3 R2 E D R R D Q E D Q Q Q R Q Q3 R2 Q H1 Q2 23768154 214650 1 43 GAC GAG CAC CTA AAG ACC GCC ACT CCG R N Q3 R N R E N E E I Q Q Q R Q Q3 R2 R Q3 R2 Q N3 Q2 E D R 32148360 235106 1 44 CCC GTC GGG TAG ACG GCG GTT ACC GCC E Q3 R2 R I R2 R H R I N R Q E R R E R R I Q3 Q Q3 R2 R H1 R2 15678117 218000 1 45 AGG CCA GGG GCC CCC TAC GCC CCA ACA Q Q R E D Q R H R R Q3 R2 E Q3 R2 I N E R Q3 R2 E D Q Q D Q 12322980 331208 1 46 CAA GCT CCC GTA ACA CAA CCC CGG AGA E N Q3 R N3 Q2 E Q3 R2 R I Q Q D Q E N Q3 E Q3 R2 E H R Q N Q 29693385 180837 1 47 CCC GGG GGC CCC CCT CCG GCG GAC GAT E Q3 R2 R H R R H1 R2 E Q3 R2 E N3 Q2 E D R R E R R N Q3 R Q Q2 21161601 339032 1 48 ACT GCC CAA ATC AAG GCC CCG AGA CAT Q N3 Q2 R Q3 R2 E N Q3 Q I R2 Q Q R R Q3 R2 E D R Q N Q E Q1 Q2 57865212 138864 1 49 ACT ACT CCC ACG CGG CGG ACA CCC AAC Q N3 Q2 Q N3 Q2 E Q3 R2 Q E R E H R E H R Q D Q E Q3 R2 Q Q1 R2 30357018 228773 1 50 ATC GAC CCG CAT GCG CTG GCG CAG ACG Q I R2 R N Q3 E D R E Q1 Q2 R E R E I R R E R E Q R Q E R 17484525 223352 1 51 AGT ACC GCG GAT CAG GCG GCG TCC GTA Q H1 Q2 Q Q3 R2 R E R R Q Q2 E Q R R E R R E R I Q3 R2 R I Q 19081170 276357 1 52 GCA GCT CAA GCA GGC GAC AAC GCC GAA R D Q R N3 Q2 E N Q3 R D Q R H1 R2 R N Q3 Q Q1 R2 R Q3 R2 R N Q 70750890 169007 1 53 GTG ACC GAC ACG CCC AGT AAG TCG GGA R I R Q Q3 R2 R N Q3 Q E R E Q3 R2 Q H1 Q2 Q Q R I D R R N Q 24010695 209162 1 54 GAC CTG GCG GCG GCC GCC GGT AGC AAG R N Q3 E I R R E R R E R R Q3 R2 R Q3 R2 R H Q2 Q H1 R2 Q Q R 28604262 232239 1 55 ACC GCC GCC GAG CTC TGC GGC GCT GAA Q Q3 R2 R Q3 R2 R Q3 R2 R N R E I R2 I1 H1 R2 R H1 R2 R N3 Q2 R N Q 70547100 74401 1 56 AAA GAC CGC AAA GCA GGG ACG CAA GAT R N Q R N Q3 E H1 R2 R N Q R D Q R H R Q E R E N Q3 R Q Q2 35291445 172547 1 57 CCT ACG GCG CTA GAC AGG ACT CCG CAG E N3 Q2 Q E R R E R E I Q R N Q3 Q Q R Q N3 Q2 E D R E Q R 199096560 280252 1 58 AGA GCG CAC CGG CAG ACG CGC GGG AAT Q N Q R E R E N E E H R E Q R Q E R E H1 R2 R H R Q N Q3 23780730 194692 1 59 GGA CGT CGC GCA GAC ACC GCC CAA AGA R N Q E H Q2 E H1 R2 R D Q R N Q3 Q Q3 R2 R Q3 R2 E N Q3 Q N Q 34512156 201230 1 60 AAT CAG TCA GAC CCA GAG GGT GAA GCT Q N Q3 E Q R I D Q R N Q3 E D Q R N R R H Q2 R N Q R N3 Q2 58798980 120510 1 61 CAA CAT CGT CCC GGC GAC ACC GAC GAC E N Q3 E Q1 Q2 E H Q2 E Q3 R2 R H1 R2 R N Q3 Q Q3 R2 R N Q3 R N Q3 43500744 201578 1 62 GGA GCC CGT GCG GCG GAT CCG CGC TAA R N Q R Q3 R2 E H Q2 R E R R E R R Q Q2 E D R E H1 R2 I N Q 18935478 228209 1 63 GCC ACT CAG GCA GCA ACT CAG GCA GCC R Q3 R2 Q N3 Q2 E Q R R D Q R D Q Q N3 Q2 E Q R R D Q R Q3 R2 49245408 236014 1 64 CCA AAT GGT CAA GCC GGG GAG GCA AAC E D Q Q N Q3 R H Q2 E N Q3 R Q3 R2 R H R R N R R D Q Q Q1 R2 72475200 122212 1 65 CCA ATG GCC ATC GAC GCG GCG ACC ACA E D Q Q I R R Q3 R2 Q I R2 R N Q3 R E R R E R Q Q3 R2 Q D Q 40564530 138662 1 66 GAC CGT CAG GAT CGC GAT TAC CGG CCA R N Q3 E H Q2 E Q R R Q Q2 E H1 R2 R Q Q2 I N E E H R E D Q 18477492 161318 1 67 ACA GCG GCA GCT ACT ACC TCT CCC GAA Q D Q R E R R D Q R N3 Q2 Q N3 Q2 Q Q3 R2 I1 N3 Q2 E Q3 R2 R N Q 57290895 157788 1 68 GCC CAG CCA ACC ACG GGG AGG GCT AGG R Q3 R2 E Q R E D Q Q Q3 R2 Q E R R H R Q Q R R N3 Q2 Q Q R 29386800 292966 1 69 AAC AAC AGG CCA GCT GGA GGG AAT ACT Q Q1 R2 Q Q1 R2 Q Q R E D Q R N3 Q2 R N Q R H R Q N Q3 Q N3 Q2 66919320 158830 1 70 AAC GCG AGC GGA ACC TAA CCG ACG CAA Q Q1 R2 R E R Q H1 R2 R N Q Q Q3 R2 I N Q E D R Q E R E N Q3 24375168 247241 1 71 GCA CGC AAC GCT GCA CGC AGC GCA GCA R D Q E H1 R2 Q Q1 R2 R N3 Q2 R D Q Q I R2 R H 2 E Q3 R2 E N Q3 28378485 203936 1 72 AAT CCG CCC ACG TAC ATC GGT CCC CAA Q N Q3 E D R E Q3 R2 Q E R I N E R H R E Q R R E R R Q3 R2 20305869 158287 1 73 GAG GTT CGG GCT TCA GGG CAG GCG GCC R N R R I Q3 E H R R N3 Q2 I D Q Q Q R E N3 Q2 R H R R I Q 21672135 188818 1 74 AAG CAG GCG CAA GGT AGG CCT GGG CTA Q Q R E Q R R E R E N Q3 R H Q2 R E R E Q1 Q2 R N R R N R 36050427 156722 1 75 CAG ACA GTA GCC TCT GCG CAT GAG GAG E Q R Q D Q R I Q R Q3 R2 I1 N3 Q2 Q Q R R Q3 R2 Q Q3 R2 R N3 Q2 29855007 211953 1 76 GCC CCC GAC CAA CTC AGG GCC ACC GCT R Q3 R2 E Q3 R2 R N Q3 E N Q3 E I R2 Q Q R R N3 Q2 Q Q1 R2 Q N Q 31503762 273280 1 77 GCT TAG GTA GCC CTG AAG GCT AAC AGA R N3 Q2 I N R R I Q R Q3 R2 E I R E D Q E H1 R2 E N Q3 E N Q 32961060 158008 1 78 GGC GAG GAC ACG GCA CCA CGC CAA CGA R H1 R2 R N R R N Q3 Q E R R D Q R H Q2 I N R I D R Q N Q 30399876 184607 1 79 ACA AGC CAC GCT CCC GGT TAG TCG AGA Q D Q Q H1 R2 E N E R N3 Q2 E Q3 R2 Q E R I I R Q Q1 R2 E Q1 Q2 22475286 148967 1 80 GAG CAA ACC GTG GCC ACG TTG AAC CAT R N R E N Q3 Q Q3 R2 R I R R Q3 R2 E H Q2 R E R R N R R N Q 50369850 79764 1 81 CCA AAC CTT ACA AGC CGT GCT GAG GAA E D Q Q Q1 R2 E I Q2 Q D Q Q H1 R2 Q Q R R N Q R N Q3 Q Q R 42618420 109734 1 82 ACT GCT GGT GCA CCG AAG GAA GAC AAG Q N3 Q2 R N3 Q2 R H Q2 R D Q E D R R Q Q2 E Q3 R2 R Q Q2 Q Q3 R2 99399690 118811 1 83 CTC CCA AGT GCG TCT GAT CCC GAT ACC E I R2 E D Q Q H1 Q2 R E R I1 N3 Q2 R I R2 R Q3 R2 E Q1 Q2 R H1 R2 30293190 147363 1 84 CAT GTA GGA AGT AAC GTC GCC CAT GGC E Q1 Q2 R I Q R N Q Q H1 Q2 Q Q1 R2 Q Q3 R2 E D Q E I R E N E 29013138 168290 1 85 GAA AAG GGC GAC GCC ACC CCA CTG CAC R N Q Q Q R R H1 R2 R N Q3 R Q3 R2 E I R2 Q Q R Q N3 Q2 Q N Q 49252356 127883 1 86 ACT CGG GGC GGT GTG CTC AGG ACT AGA Q N3 Q2 E H R R H1 R2 R H Q2 R I R E N E R Q3 R2 E Q3 R2 Q N Q3 22559176 151162 1 87 ACA CGC CAG ACG GCC CAC GCC CCC AAT Q D Q E H1 R2 E Q R Q E R R Q3 R2 I1 H1 R2 R Q Q2 E Q1 Q2 R N3 Q2 22770018 332728 1 88 AAT GGT GCT TAT GTC TGC GAT CAT GCT Q N Q3 R H Q2 R N3 Q2 I N Q3 R I R2 R H Q2 E H R Q I Q3 Q E R 36599346 105408 1 89 ACA TAT ACG GAC GGG GGT CGG ATT ACG Q D Q I N Q3 Q E R R N Q3 R H R I H R E N3 Q2 Q Q R R N Q3 21505752 166494 1 90 GAT CAT AAC CCG GGT TGG CCT AAG GAC R Q Q2 E Q1 Q2 Q Q1 R2 E D R R H Q2 Q D Q E Q R E N3 Q2 Q Q1 R2 21718140 261012 1 91 GAT CAG GCC GTT AAG ACA CAG CCT AAC R Q Q2 E 1 R R Q3 R2 R I Q3 Q Q R E Q3 R2 R N3 Q2 R Q Q2 R N3 Q2 30361419 297740 1 92 AAT GCA ACG GAT GCT CCC GCT GAT GCT Q N Q3 R D Q Q E R R Q Q2 R N3 Q2 Q I R2 Q Q R R N R Q I R 68828940 208472 1 93 GAT CCT ATC GGT GAT ATC AGG GAG ATG R Q Q2 E N3 Q2 Q I R2 R H Q2 R Q Q2 I N Q R H R Q N Q3 Q E R 41422185 110016 1 94 CAC TCT GAG GTC GGA TAA GGG AAT ACG E N E I1 N3 Q2 R N R R I R2 R N Q R E R I Q3 R2 R N Q R E R 32161923 113354 1 95 GCT CCC AGC GCA GCT GCG TCC GGA GCG R N3 Q2 E Q3 R2 Q H1 R2 R D Q R N3 Q2 I N E R D Q Q I R Q D Q 23772042 285203 1 96 GAT GGT CCC GGG CCT TAC GCA ATG ACA R Q Q2 R H Q2 E Q3 R2 R H R E N3 Q2 E D R E H1 R2 R N Q3 R Q Q2 23602050 141855 1 97 GAG ATG CGT ATC TCT CCG CGC GAC GAT R N R Q I R E H Q2 Q I R2 I1 N3 Q2 R D Q R H Q2 R N Q R Q3 R2 24759504 117310 1 98 ACT GAG GAG CCT AAG CCA GGT GAA GCC Q N3 Q2 R N R R N R E N3 Q2 Q Q R R I Q R H1 R2 Q N3 Q2 Q Q3 R2 76718610 17311796 1 99 AAC TAT GAC CCG AGG GTA GGC ACT ACC Q Q1 R2 I N Q3 R N Q3 R E R Q Q R R N Q R H1 R2 Q N3 Q2 Q Q3 R2 32201523 202169 1 100 ACT AAG CGT ACA AAG CTC GGA GCA ACC Q NE Q2 Q Q R E H Q2 Q D Q Q Q R E I R2 R N Q R D Q Q Q3 R2 50819418 124234 *The Optimal DNA sequence in gene 1 is shown as SEQ ID NO: 109 in the Sequence Listing. The sequences in genes 3-31 are shown as SEQ ID NOS: 110-138, respectively. The sequences in genes 33-100 are shown as SEQ ID NOS: 139-206, respectively.

REFERENCES

1. Miller, J., McLachlan, A. D. and Klug, A. (1985) EMBO J. 4, 1609-1614.

2. Berg, J. M. (1988) Proc. Natl. Acad. Sci. USA 85, 99-102.

3. Gibson, T. J., Postma, J. P. M., Brown, R. S. and Argos, P. (1988) Protein Eng. 2, 209-218.

4. Lee, M. S., Gippert, G. P., Soman, K. V., Case, D. A. and Wright, P. E. (1989) Science 245, 635-637.

5. Nardelli, J., Gibson, T. J., Vesque, C. and Charnay, P. (1991) Nature 349, 175-178.

6. Thiesen, H. J. and Bach, C. (1991) FEBS Lett. 283, 23-26.

7. Pavletich, N. P. and Pabo, C. O. (1991) Science 252, 809-817.

8. Berg, J. M. (1992) Proc. Natl. Acad. Sci. USA89, 11109-11110.

9. Kriwacki, R. W., Schultz, S. C., Steitz, T. A. and Caradonna, J. P. (1992) Proc. Natl. Acad. Sci. USA 89, 9759-9763.

10. Jacobs, G. H. (1992) EMBO J. 11, 4507-4517.

11. Gogos, J. A., Hsu, T., Bolton, J. and Kafatos, F. C. (1992) Science 257, 1951-1955.

12. Fairall, L., Harrison, S. D., Travers, A. A. and Rhodes, D. (1992) J. Mol. Biol. 226, 349-366.

13. Klevit, R. E. (1991) Science 253, 1367 and 1393.

14. Desjarlais, J. R. and Berg, J. M. (1992) Proc. Natl. Acad. Sci. USA 89, 7345-7349.

15. Rebar, E. J., and Pabo, C. O., U.S. Pat. No. 5,789,538, Aug. 4, 1998.

16. Beerli, R. R., Segal, D. J., Dreier, B., and Barbas, C. F. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 14628-14633.

17. Jordan, S. R. and Pabo, C. O. (1988) Science 242, 893-899.

18. Aggarwal, A. K., Rodgers, D. W., Drottar, M., Ptashne, M. and Harrison S. C. (1988) Science 242, 899-907.

19. Letovsky, J., Dyran, W. S. (1989) Nucleic Acids Res. 17, 2639-2653.

20. Kissinger, C. R., Liu, B., Martin-Blanco, E., Kornberg, T. B. and Pabo, C. O. (1990) Cell 63, 579-590.

21. Wolberger, C., Vershon, A. K., Liu, B., Johnson, A. D. and Pabo, C. O. (1991) Cell 67, 517-528.

22. Seeman, N.C., Rosenberg, J. M. and Rich, A. (1976) Proc. Natl. Acad. Sci USA 73, 804-808.

23. Mikelsaar, R. -H., Bruskov, V. I. and Poltev, V. I. (1985) New precision space-filling atomic-molecular models, Pushchino.

24. Mikelsaar, R. (1986) Trends in Biotechnology 4, 162-163.

231 1 34 PRT Artificial Sequence Description of Artificial Sequence Hypothetical Zinc-Finger Binding Site 1 Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Leu Xaa Xaa His Xaa Xaa Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Glu 20 25 30 Xaa Pro 2 8 PRT Artificial Sequence Description of Artificial Sequence Zinc-Finger Domain Segment 2 Xaa Xaa Xaa Xaa Leu Xaa Xaa His 1 5 3 27 PRT Artificial Sequence Description of Artificial Sequence Zinc-Finger Domain Segment 3 Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Leu Xaa Xaa His Xaa Xaa Xaa Xaa Xaa His 20 25 4 7 PRT Artificial Sequence Description of Artificial Sequence Peptide Linker 4 Xaa Xaa Xaa Xaa Glu Xaa Pro 1 5 5 27 DNA Saccharomyces cerevisiae 5 cctactctca gattccactt cactcca 27 6 27 DNA Saccharomyces cerevisiae 6 tcagagctca cttagcccaa tactaca 27 7 27 DNA Saccharomyces cerevisiae 7 gccgcttggg acgtcgcaga aggagcc 27 8 27 DNA Saccharomyces cerevisiae 8 gaaaagcata cagcttccga tacatca 27 9 27 DNA Saccharomyces cerevisiae 9 agtagcataa taggatctag tactgcc 27 10 27 DNA Saccharomyces cerevisiae 10 gccgtggaaa caattgcaga ggagatg 27 11 27 DNA Saccharomyces cerevisiae 11 ccaagtcgag acgcttaaag ttaagtg 27 12 27 DNA Saccharomyces cerevisiae 12 gactacagcg gcatgcctgc cagcaaa 27 13 27 DNA Saccharomyces cerevisiae 13 acttggactg ccgagcgggt cgatcaa 27 14 27 DNA Saccharomyces cerevisiae 14 gacccaactg cgcacgctgc caaggag 27 15 27 DNA Saccharomyces cerevisiae 15 gctgccagca gttgtgcgga cctgcat 27 16 27 DNA Saccharomyces cerevisiae 16 gataacacag tgcagaagac tcctaca 27 17 27 DNA Saccharomyces cerevisiae 17 tatgcacccg atacagaggg tgtcgag 27 18 27 DNA Saccharomyces cerevisiae 18 caagagcacg acggccgcag taagcac 27 19 27 DNA Saccharomyces cerevisiae 19 gagatacccg tgatacgtcg cggtaaa 27 20 27 DNA Saccharomyces cerevisiae 20 aatggggcac gacctccaga gggtgat 27 21 27 DNA Saccharomyces cerevisiae 21 gagagacccc aggcgtgaga cacgtct 27 22 27 DNA Saccharomyces cerevisiae 22 acctccgctg tcatgggtac gggtggc 27 23 27 DNA Saccharomyces cerevisiae 23 gaaccgagtt aggggcgatc taagcga 27 24 27 DNA Saccharomyces cerevisiae 24 gatggaacac ccaagggtcg tcgtgaa 27 25 27 DNA Saccharomyces cerevisiae 25 accgaccata caggacgtgc tgccacc 27 26 27 DNA Saccharomyces cerevisiae 26 gaaagacgga agctcgatgc taacgct 27 27 27 DNA Saccharomyces cerevisiae 27 agtcaactgg gaagggtccg gcatcat 27 28 27 DNA Saccharomyces cerevisiae 28 aggatggtac gtgccacggg accatct 27 29 27 DNA Saccharomyces cerevisiae 29 atggaccacc cgcatgcccg ctgtgag 27 30 27 DNA Saccharomyces cerevisiae 30 aagaccccca cgcctgtgtc cgcacct 27 31 27 DNA Saccharomyces cerevisiae 31 gatcagcccg ccggccctgg tgcggac 27 32 27 DNA Saccharomyces cerevisiae 32 agtccgcacc agggccggcg ggctgat 27 33 27 DNA Saccharomyces cerevisiae 33 aatggcgcta accgggacag tagcgac 27 34 27 DNA Saccharomyces cerevisiae 34 gccactgccg cctctgtcag cgctgcc 27 35 27 DNA Saccharomyces cerevisiae 35 actccgaggc gagcgcaaag cggaaca 27 36 27 DNA Saccharomyces cerevisiae 36 accgtcgctg cctccgctgt cgctgct 27 37 27 DNA Saccharomyces cerevisiae 37 acgtaggcag acgagctcag tcaagag 27 38 27 DNA Saccharomyces cerevisiae 38 accaccctgg ttaccgtccc cggtgtc 27 39 27 DNA Saccharomyces cerevisiae 39 acaacccagg aaaacgcctc cgaagcc 27 40 27 DNA Saccharomyces cerevisiae 40 caacaagcga gatgggcgga tatccca 27 41 27 DNA Saccharomyces cerevisiae 41 gccggatgcg aggacgcaag cagggga 27 42 27 DNA Saccharomyces cerevisiae 42 aaaggtaccg ctacgccacc tacgggt 27 43 27 DNA Saccharomyces cerevisiae 43 actgatcgtg aaccccgtca aggtaag 27 44 27 DNA Saccharomyces cerevisiae 44 caacgggact ccgcagacgg gagcaat 27 45 27 DNA Saccharomyces cerevisiae 45 cccgaggagg tacccctaga tcactat 27 46 27 DNA Saccharomyces cerevisiae 46 gacccttatg ctctatccga gcacgat 27 47 27 DNA Saccharomyces cerevisiae 47 caaggtggac agccgaacat agctggt 27 48 27 DNA Saccharomyces cerevisiae 48 ccggattaca cgtctgcctc gaccgca 27 49 27 DNA Saccharomyces cerevisiae 49 ggtgccgata cggataatgc ggtaact 27 50 27 DNA Saccharomyces cerevisiae 50 atcagcgact ctaggccgca cgttcag 27 51 27 DNA Saccharomyces cerevisiae 51 acgcctgaag aggcgcacac tcctgcc 27 52 27 DNA Saccharomyces cerevisiae 52 tctagtgccc ggaacacacg gagagca 27 53 27 DNA Saccharomyces cerevisiae 53 agcgctgatg agagagacgc ggaagat 27 54 27 DNA Saccharomyces cerevisiae 54 accgccgcac caacggcact cgcgacg 27 55 27 DNA Saccharomyces cerevisiae 55 acgggagata gcactccctc aggcacg 27 56 27 DNA Saccharomyces cerevisiae 56 gacgagggcg gccgcatagt gcacgca 27 57 27 DNA Saccharomyces cerevisiae 57 accgctggcg cagacgccac tagcaag 27 58 27 DNA Saccharomyces cerevisiae 58 tatgagccgt accagatacg tgctaat 27 59 27 DNA Saccharomyces cerevisiae 59 cagacaccac cgagccccga tcaagag 27 60 27 DNA Saccharomyces cerevisiae 60 catcgcgttg gcactcggtc ccgaaag 27 61 27 DNA Saccharomyces cerevisiae 61 gctattgggc ctgcccggtg tagggcc 27 62 27 DNA Saccharomyces cerevisiae 62 attcctgata cggcggttga caaggag 27 63 27 DNA Saccharomyces cerevisiae 63 gacgccaggg acgagcaagg ggacgaa 27 64 27 DNA Saccharomyces cerevisiae 64 gtcgccaacg ctcacggtgt ggaaacc 27 65 27 DNA Saccharomyces cerevisiae 65 agaaggggca agcgtggtcg tgacgat 27 66 27 DNA Saccharomyces cerevisiae 66 cagcagagcg actaaactac tgcacga 27 67 27 DNA Saccharomyces cerevisiae 67 gatgtgggga accatgccca ggatgat 27 68 27 DNA Saccharomyces cerevisiae 68 actgtgggca acagtacggc aattacc 27 69 27 DNA Saccharomyces cerevisiae 69 actgacgagc atgaagctga tgtcaat 27 70 27 DNA Saccharomyces cerevisiae 70 gacccagaag tgcaggctga catgaag 27 71 27 DNA Saccharomyces cerevisiae 71 gatcattagc aaagtgagca acacacg 27 72 27 DNA Saccharomyces cerevisiae 72 gatgggaagt aagacccccg atatgag 27 73 27 DNA Saccharomyces cerevisiae 73 ggtgctcggc atacagccct gattgtt 27 74 27 DNA Saccharomyces cerevisiae 74 catgtcagca ctgcacagga gccgaaa 27 75 27 DNA Saccharomyces cerevisiae 75 gctagcgcta ctgtgccttc cgctccc 27 76 27 DNA Saccharomyces cerevisiae 76 gatcgtgaag ctacctttag agacgcc 27 77 27 DNA Saccharomyces cerevisiae 77 aacgttggtg agagtcctgg aaggggc 27 78 27 DNA Saccharomyces cerevisiae 78 gctgaagccg actatcttgc cgatgag 27 79 27 DNA Saccharomyces cerevisiae 79 gaataacatt agagcggggc gccaaac 27 80 27 DNA Saccharomyces cerevisiae 80 caatgcacag acaaattcga tactcac 27 81 27 DNA Saccharomyces cerevisiae 81 gctaatcacg tcgacgtcgg gcatgga 27 82 27 DNA Saccharomyces cerevisiae 82 cctgcccagg ccgctgagct ggtgggt 27 83 27 DNA Saccharomyces cerevisiae 83 acccaggcag tggaggagcg agccagg 27 84 27 DNA Saccharomyces cerevisiae 84 cgagcggctc ccgctccctg ggcagaa 27 85 27 DNA Saccharomyces cerevisiae 85 gtgcctccgc cgcagcaacc actacat 27 86 27 DNA Saccharomyces cerevisiae 86 cctgatgtac agtgggatac agcgaat 27 87 27 DNA Saccharomyces cerevisiae 87 ccaacgaacg gtgcagagaa tacgcag 27 88 27 DNA Saccharomyces cerevisiae 88 ggtgatcgca cgcaagcctg cggggaa 27 89 27 DNA Saccharomyces cerevisiae 89 gagaagccaa caagcaacgc tgaggaa 27 90 27 DNA Saccharomyces cerevisiae 90 gaggcgacat aagttgttag cacagca 27 91 27 DNA Saccharomyces cerevisiae 91 gatcatgggc tatgggatac tccctac 27 92 27 DNA Saccharomyces cerevisiae 92 aagttagata cacttaacga acctagt 27 93 27 DNA Saccharomyces cerevisiae 93 aatgctccca ggggctcggc caggacc 27 94 27 DNA Saccharomyces cerevisiae 94 gattcgccac ggcacactcc gcacaga 27 95 27 DNA Saccharomyces cerevisiae 95 gcggagtgag ccgccgaggc caacgac 27 96 27 DNA Saccharomyces cerevisiae 96 acatcgccgc ctaccagtcc cgcggtg 27 97 27 DNA Saccharomyces cerevisiae 97 ggtatcgcca gtctccgggt ccatacc 27 98 27 DNA Saccharomyces cerevisiae 98 atcactcccg gtccacattc taggaag 27 99 27 DNA Saccharomyces cerevisiae 99 aatgtcagtt gtgacgctag acttttc 27 100 27 DNA Saccharomyces cerevisiae 100 aatactgtag ctgctgagac gattacc 27 101 27 DNA Saccharomyces cerevisiae 101 agctgtggaa acaatcgcag gggagat 27 102 27 DNA Saccharomyces cerevisiae 102 gtagaaatct gctgcacatg ccacacg 27 103 27 DNA Saccharomyces cerevisiae 103 gctacggaga aaccgtgcac taactct 27 104 27 DNA Saccharomyces cerevisiae 104 accgtttcct ccaagtcata caccact 27 105 27 DNA Saccharomyces cerevisiae 105 ctatatattt gtggcagaaa tcatacc 27 106 27 DNA Saccharomyces cerevisiae 106 tatcatgaca acatggtaca aattgag 27 107 27 DNA Saccharomyces cerevisiae 107 aagaccgcac tagatcttac caagagc 27 108 27 DNA Saccharomyces cerevisiae 108 atgacgacat ccaagccagc ttttacc 27 109 27 DNA Caenorhabditis elegans 109 acgaagccgc ccgactccta ggagatg 27 110 27 DNA Caenorhabditis elegans 110 gacgccgagc acgagcacag gagcgaa 27 111 27 DNA Caenorhabditis elegans 111 gacaattctc ccggcgccaa tgatcac 27 112 27 DNA Caenorhabditis elegans 112 agcacgactc ggcacacccg ttccgcc 27 113 27 DNA Caenorhabditis elegans 113 ccaggaaggc gcactggcag acgtaag 27 114 27 DNA Caenorhabditis elegans 114 gctattactc gtggtgccgc gggagcc 27 115 27 DNA Caenorhabditis elegans 115 aatagcgaac agatatctga caactcc 27 116 27 DNA Caenorhabditis elegans 116 gtcgcgcata ctaaggctcg ctttaat 27 117 27 DNA Caenorhabditis elegans 117 gatattcgcg atggcaccgg tgacgat 27 118 27 DNA Caenorhabditis elegans 118 cataacgtcg aggctgcccg caaggag 27 119 27 DNA Caenorhabditis elegans 119 accagccatc acgccatgcg aagcacc 27 120 27 DNA Caenorhabditis elegans 120 aggcaccccg ggaagcgccg gaccgaa 27 121 27 DNA Caenorhabditis elegans 121 ggagcaccag acgccccgac taagccg 27 122 27 DNA Caenorhabditis elegans 122 gatgctcgcc tactcgcgag gccaaga 27 123 27 DNA Caenorhabditis elegans 123 agagccgatg cccgcaccaa ggctgct 27 124 27 DNA Caenorhabditis elegans 124 acagacgagg ccaagatcta cgccgaa 27 125 27 DNA Caenorhabditis elegans 125 ccaaccggta ggccgatagg tagtcgc 27 126 27 DNA Caenorhabditis elegans 126 cctacaaggc cccgtacgag ggccgat 27 127 27 DNA Caenorhabditis elegans 127 accaacggac aggatggcgc cgaacaa 27 128 27 DNA Caenorhabditis elegans 128 gcaagccgct atgtcgacgc tcgccaa 27 129 27 DNA Caenorhabditis elegans 129 agtccgccta agcgccctgt tccgccg 27 130 27 DNA Caenorhabditis elegans 130 aagcggatcg ctgcgaccga accgcct 27 131 27 DNA Caenorhabditis elegans 131 aatgatctag ctcccagtcc tactgcg 27 132 27 DNA Caenorhabditis elegans 132 cagaggactc gccgtatcgt cgctggt 27 133 27 DNA Caenorhabditis elegans 133 gctatgaggt cacgggctcc ccctgat 27 134 27 DNA Caenorhabditis elegans 134 gacacaggtc cccatcacgg gacaagt 27 135 27 DNA Caenorhabditis elegans 135 cacgttggga ccgccaggag cccgaat 27 136 27 DNA Caenorhabditis elegans 136 cgagaacgtc ccatcaagcg tgaacac 27 137 27 DNA Caenorhabditis elegans 137 gatccttcga gtacgcctac gcaagga 27 138 27 DNA Caenorhabditis elegans 138 aacgccgcct agaccccggg gaatacc 27 139 27 DNA Caenorhabditis elegans 139 gtggtgcagg aggggcagga ggccaca 27 140 27 DNA Caenorhabditis elegans 140 cgtacaagga ataccctgaa ggacaac 27 141 27 DNA Caenorhabditis elegans 141 acctccactc agcgaccacc ggcgcca 27 142 27 DNA Caenorhabditis elegans 142 aaagcggtac aagcgacgac ccgtgct 27 143 27 DNA Caenorhabditis elegans 143 acaacgaccg aggagcctac cgaagcc 27 144 27 DNA Caenorhabditis elegans 144 gagaaccgtc cccacccgaa gctggct 27 145 27 DNA Caenorhabditis elegans 145 cgtcgggtgc ccgagactgg gaccggt 27 146 27 DNA Caenorhabditis elegans 146 agggaccgta cggaacagga cccccct 27 147 27 DNA Caenorhabditis elegans 147 aggcaaccct ggaggcgtcc cagcacc 27 148 27 DNA Caenorhabditis elegans 148 ctcactaccc cggcaccaag gaccagt 27 149 27 DNA Caenorhabditis elegans 149 gacgagcacc taaagaccgc cactccg 27 150 27 DNA Caenorhabditis elegans 150 cccgtcgggt agacggcggt taccggc 27 151 27 DNA Caenorhabditis elegans 151 aggccagggg ccccctacgc cccaaca 27 152 27 DNA Caenorhabditis elegans 152 caagctcccg taacacaacc ccggaga 27 153 27 DNA Caenorhabditis elegans 153 cccgggggcc cccctccggc ggacgat 27 154 27 DNA Caenorhabditis elegans 154 actgcccaaa tcaaggcccc gagacat 27 155 27 DNA Caenorhabditis elegans 155 actactccca cgcggcggac acccaac 27 156 27 DNA Caenorhabditis elegans 156 atcgacccgc atgcgctggc gcagacg 27 157 27 DNA Caenorhabditis elegans 157 agtaccgcgg atcaggcggc gtccgta 27 158 27 DNA Caenorhabditis elegans 158 gcagctcaag caggcgacaa cgccgaa 27 159 27 DNA Caenorhabditis elegans 159 gtgaccgaca cgcccagtaa gtcggga 27 160 27 DNA Caenorhabditis elegans 160 gacctggcgg cggccgccgg tagcaag 27 161 27 DNA Caenorhabditis elegans 161 accgccgccg agctctgcgg cgctgaa 27 162 27 DNA Caenorhabditis elegans 162 aaagaccgca aagcagggac gcaagat 27 163 27 DNA Caenorhabditis elegans 163 cctacggcgc tagacaggac tccgcag 27 164 27 DNA Caenorhabditis elegans 164 agagcgcacc ggcagacgcg cgggaat 27 165 27 DNA Caenorhabditis elegans 165 ggacgtcgcg cagacaccgc ccaaaga 27 166 27 DNA Caenorhabditis elegans 166 aatcagtcag acccagaggg tgaagct 27 167 27 DNA Caenorhabditis elegans 167 caacatcgtc ccggcgacac cgacgac 27 168 27 DNA Caenorhabditis elegans 168 ggagcccgtg cggcggatcc gcgctaa 27 169 27 DNA Caenorhabditis elegans 169 gccactcagg cagcaactca ggcagcc 27 170 27 DNA Caenorhabditis elegans 170 ccaaatggtc aagccgggga ggcaaac 27 171 27 DNA Caenorhabditis elegans 171 ccaatggcca tcgacgcggc gaccaca 27 172 27 DNA Caenorhabditis elegans 172 gaccgtcagg atcgcgatta ccggcca 27 173 27 DNA Caenorhabditis elegans 173 acagcggcag ctactacctc tcccgaa 27 174 27 DNA Caenorhabditis elegans 174 gcccagccaa ccacggggag ggctagg 27 175 27 DNA Caenorhabditis elegans 175 aacaacaggc cagctggagg gaatact 27 176 27 DNA Caenorhabditis elegans 176 aacgcgagcg gaacctaacc gacgcaa 27 177 27 DNA Caenorhabditis elegans 177 gcacgcaacg ctgcacgcag cgcagca 27 178 27 DNA Caenorhabditis elegans 178 aatccgccca cgtacatcgg tccccaa 27 179 27 DNA Caenorhabditis elegans 179 gaggttcggg cttcagggca ggcggcc 27 180 27 DNA Caenorhabditis elegans 180 aagcaggcgc aaggtaggcc tggggta 27 181 27 DNA Caenorhabditis elegans 181 cagacagtag cctctgcgca tgaggag 27 182 27 DNA Caenorhabditis elegans 182 gcccccgacc aactcagggc caccgct 27 183 27 DNA Caenorhabditis elegans 183 gcttaggtag ccctgaaggc taacaga 27 184 27 DNA Caenorhabditis elegans 184 ggcgaggaca cggcaccacg ccaacga 27 185 27 DNA Caenorhabditis elegans 185 acaagccacg ctcccggtta gtcgaga 27 186 27 DNA Caenorhabditis elegans 186 gagcaaaccg tggccacgtt gaaccat 27 187 27 DNA Caenorhabditis elegans 187 ccaaacctta caagccgtgc ggaggaa 27 188 27 DNA Caenorhabditis elegans 188 actgctggtg caccgaagga agacaag 27 189 27 DNA Caenorhabditis elegans 189 ctcccaagtg cgtctgatcc cgatacc 27 190 27 DNA Caenorhabditis elegans 190 catgtaggaa gtaacgtcgc ccatggc 27 191 27 DNA Caenorhabditis elegans 191 gaaaagggcg acgccacccc actgcac 27 192 27 DNA Caenorhabditis elegans 192 actcggggcg gtgtgctcag gactaga 27 193 27 DNA Caenorhabditis elegans 193 acacgccaga cggcccacgc ccccaat 27 194 27 DNA Caenorhabditis elegans 194 aatggtgctt atgtctgcga tcatgct 27 195 27 DNA Caenorhabditis elegans 195 acatatacgg acgggggtcg gattacg 27 196 27 DNA Caenorhabditis elegans 196 gatcataacc cgggttggcc taaggac 27 197 27 DNA Caenorhabditis elegans 197 gatcaggccg ttaagacaca gcctaac 27 198 27 DNA Caenorhabditis elegans 198 aatgcaacgg atgctcccgc tgatgct 27 199 27 DNA Caenorhabditis elegans 199 gatcctatcg gtgatatcag ggagatg 27 200 27 DNA Caenorhabditis elegans 200 cactctgagg tcggataagg gaatacg 27 201 27 DNA Caenorhabditis elegans 201 gctcccagcg cagctgcgtc cggagcg 27 202 27 DNA Caenorhabditis elegans 202 gatggtcccg ggccttacgc aatgaca 27 203 27 DNA Caenorhabditis elegans 203 gagatgcgta tctctccgcg cgacgat 27 204 27 DNA Caenorhabditis elegans 204 actgaggagc ctaaggcagg tgaagcc 27 205 27 DNA Caenorhabditis elegans 205 aactatgacg cgagggtagg cactacc 27 206 27 DNA Caenorhabditis elegans 206 actaagcgta caaagctcgg agcaacc 27 207 25 PRT Xenopus laevis 207 Tyr Ile Cys Ser Phe Ala Asp Cys Gly Ala Ala Tyr Asn Lys Asn Trp 1 5 10 15 Lys Leu Gln Ala His Leu Cys Lys His 20 25 208 30 PRT Xenopus laevis 208 Thr Gly Glu Lys Pro Phe Pro Cys Lys Glu Glu Gly Cys Glu Lys Gly 1 5 10 15 Phe Thr Ser Leu His His Leu Thr Arg His Ser Leu Thr His 20 25 30 209 31 PRT Xenopus laevis 209 Thr Gly Glu Lys Asn Phe Thr Cys Asp Ser Asp Gly Cys Asp Leu Arg 1 5 10 15 Phe Thr Thr Lys Ala Asn Met Lys Lys His Phe Asn Arg Phe His 20 25 30 210 31 PRT Xenopus laevis 210 Asn Ile Lys Ile Cys Val Tyr Val Cys His Phe Glu Asn Cys Gly Lys 1 5 10 15 Ala Phe Lys Lys His Asn Gln Leu Lys Val His Gln Phe Ser His 20 25 30 211 30 PRT Xenopus laevis 211 Thr Gln Gln Leu Pro Tyr Glu Cys Pro His Glu Gly Cys Asp Lys Arg 1 5 10 15 Phe Ser Leu Pro Ser Arg Leu Lys Arg His Glu Lys Val His 20 25 30 212 29 PRT Xenopus laevis 212 Ala Gly Tyr Pro Cys Lys Lys Asp Asp Ser Cys Ser Phe Val Gly Lys 1 5 10 15 Thr Trp Thr Leu Tyr Leu Lys His Val Ala Glu Cys His 20 25 213 26 PRT Xenopus laevis 213 Gln Asp Leu Ala Val Cys Asp Val Cys Asn Arg Lys Phe Arg His Lys 1 5 10 15 Asp Tyr Leu Arg Asp His Gln Lys Thr His 20 25 214 32 PRT Xenopus laevis 214 Glu Lys Glu Arg Thr Val Tyr Leu Cys Pro Arg Asp Gly Cys Asp Arg 1 5 10 15 Ser Tyr Thr Thr Ala Phe Asn Leu Arg Ser His Ile Gln Ser Phe His 20 25 30 215 30 PRT Xenopus laevis 215 Glu Glu Gln Arg Pro Phe Val Cys Glu His Ala Gly Cys Gly Lys Cys 1 5 10 15 Phe Ala Met Lys Lys Ser Leu Glu Arg His Ser Val Val His 20 25 30 216 25 PRT Xenopus laevis 216 Tyr Lys Cys Gly Leu Cys Glu Arg Ser Phe Val Glu Lys Ser Ala Leu 1 5 10 15 Ser Arg His Gln Arg Val His Lys Asn 20 25 217 29 PRT Saccharomyces cerevisiae 217 Thr Asn Leu Lys Pro Tyr Pro Cys Gly Leu Cys Asn Arg Cys Phe Thr 1 5 10 15 Arg Arg Asp Leu Leu Ile Arg His Ala Gln Lys Ile His 20 25 218 23 PRT Mus musculus 218 Tyr Gly Cys Asp Glu Cys Gly Lys Thr Phe Arg Gln Ser Ser Ser Leu 1 5 10 15 Leu Lys His Gln Arg Ile His 20 219 28 PRT Mus musculus 219 Thr Gly Glu Lys Pro Tyr Thr Cys Asn Val Cys Asp Lys His Phe Ile 1 5 10 15 Glu Arg Ser Ser Leu Thr Val His Gln Arg Thr His 20 25 220 28 PRT Mus musculus 220 Thr Gly Glu Lys Pro Tyr Lys Cys His Glu Cys Gly Lys Ala Phe Ser 1 5 10 15 Gln Ser Met Asn Leu Thr Val His Gln Arg Thr His 20 25 221 28 PRT Mus musculus 221 Thr Gly Glu Lys Pro Tyr Gln Cys Lys Glu Cys Gly Lys Ala Phe Arg 1 5 10 15 Lys Asn Ser Ser Leu Ile Gln His Glu Arg Ile His 20 25 222 28 PRT Mus musculus 222 Thr Gly Glu Lys Pro Tyr Lys Cys His Asp Cys Glu Lys Ala Phe Ser 1 5 10 15 Lys Asn Ser Ser Leu Thr Gln His Arg Arg Ile His 20 25 223 28 PRT Mus musculus 223 Thr Gly Glu Lys Pro Tyr Glu Cys Met Ile Cys Gly Lys His Phe Thr 1 5 10 15 Gly Arg Ser Ser Leu Thr Val His Gln Val Ile His 20 25 224 28 PRT Mus musculus 224 Thr Gly Glu Lys Pro Tyr Glu Cys Thr Glu Cys Gly Lys Ala Phe Ser 1 5 10 15 Gln Ser Ala Tyr Leu Ile Glu His Arg Arg Ile His 20 25 225 28 PRT Mus musculus 225 Thr Gly Glu Lys Pro Tyr Glu Cys Asp Gln Cys Gly Lys Ala Phe Ile 1 5 10 15 Lys Asn Ser Ser Leu Ile Val His Gln Arg Ile His 20 25 226 28 PRT Mus musculus 226 Thr Gly Glu Lys Pro Tyr Gln Cys Asn Glu Cys Gly Lys Pro Phe Ser 1 5 10 15 Arg Ser Thr Asn Leu Thr Arg His Gln Arg Thr His 20 25 227 23 PRT Drosophila melanogaster 227 Phe Thr Cys Lys Ile Cys Ser Arg Ser Phe Gly Tyr Lys His Val Leu 1 5 10 15 Gln Asn His Glu Arg Thr His 20 228 28 PRT Drosophila melanogaster 228 Thr Gly Glu Lys Pro Phe Glu Cys Pro Glu Cys Asp Lys Arg Phe Thr 1 5 10 15 Arg Asp His His Leu Lys Thr His Met Arg Leu His 20 25 229 28 PRT Drosophila melanogaster 229 Thr Gly Glu Lys Pro Tyr His Cys Ser His Cys Asp Arg Gln Phe Val 1 5 10 15 Gln Val Ala Asn Leu Arg Arg His Leu Arg Val His 20 25 230 28 PRT Drosophila melanogaster 230 Thr Gly Glu Lys Pro Tyr Thr Cys Glu Ile Cys Asp Gly Lys Phe Ser 1 5 10 15 Asp Ser Asn Gln Leu Lys Ser His Met Leu Val His 20 25 231 5 PRT Drosophila melanogaster 231 Thr Gly Glu Lys Pro 1 5 

What is claimed is:
 1. A method for designing a DNA-binding protein (DBP), with multiple zinc-finger (ZF) domains connected by linker sequences, that binds selectively to a target DNA sequence within a given gene, each of said ZF domains having the formula A₁XCX₂₋₄CA₂A₃XFXZ₃XXZ₂LXZ₁HX₃₋₅H   (SEQ ID NO: 3) and each of said linkers having the formula A₄A₅X₀₋₂EA₆ P   (SEQ ID NO 4), wherein (i) X is any amino acid; (ii) X₂₋₄ is a peptide from 2 to 4 amino acids in length; (iii) X₃₋₅ is a peptide from 3 to 5 amino acids in length; (iv) X₀₋₂ is a peptide from 0 to 2 amino acids in length; (iv) A₁ is selected from the group consisting of phenylalanine and tyrosine; (v) A₂ is selected from the group consisting of glycine and aspartic acid; (vi) A₃ is selected from the group consisting of lysine and arginine; (vii) A₄ is selected from the group consisting of threonine and serine; (viii) A₅ is selected from the group consisting of glycine and glutamic acid; (ix) A₆ is selected from the group consisting of lysine and arginine; (x) C is cysteine; (xi) F is phenylalanine; (xii) L is leucine; (xiii) H is histidine; (xiv) E is glutamic acid; (xv) P is proline; and (xvi) Z₁, Z₂ and Z₃ are the base-contacting amino acids, which method comprises an algorithm comprising the steps of: (a) setting a genome to be screened; (b) selecting the target DNA sequence in the genome for binding; (c) setting the number of ZF domains to n_(d); (d) dividing the target DNA sequence into nucleotide blocks wherein each block contains n_(z) nucleotides using a first routine where n_(z) is determined using the following relationship: n_(z)=3n_(d); (e) assigning base-contacting amino acids at Z₁, Z₂ and Z₃ to each ZF domain, according to the A Rules and/or B Rules set forth in Tables 1-3 of the specification, of a DBP which binds to the first nucleotide block from step (d) as numbered from the first 5′ nucleotide of the target gene sequence to generate a block-specific DBP and calculating the binding energy, Binding Energy_(block), of each ZF domain of each such block-specific DBP as the product of the binding energies, Binding Energy_(domain), of all ZF domains of the DBP, each determined using the formula: Binding Energy_(domain)=(5×the number of hydrogen bonds)+(2×the number of H₂O contacts)+(the number of hydrophobic contacts); (f) subdividing the DBP from step (d) into blocks using a second routine to generate a subdivided DBP having three ZF domains; (g) screening the subdivided DBP from step (f) against the genome using a third routine to determine the number of binding sites in the genome for each subdivided DBP in the genome and assigning a binding energy for each such site using the following formula: Binding Energy_(site n)=(5×the number of hydrogen bonds)+(2×the number of H₂O contacts)+(the number of hydrophobic contacts); (h) calculating a ratio of binding energy, R_(b), using a fourth routine for each nucleotide block-specific DBP from step (e) using the following formula: R_(b)=Binding Energy_(block)/the sum of all Binding Energy_(site n)'s for all subdivided DBP's from step (g); (i) repeating steps (f) through (h) for each subdivided DBP wherein n_(d)≧4; (j) repeating steps (d) through (i) for each nucleotide block in the target DNA sequence containing n_(z) nucleotides; (k) rank-ordering R_(b) numerical values obtained from step (h); and (l) selecting a DBP with an acceptable R_(b) value.
 2. The method of claim 1 wherein the DBP selected is that whose R_(b) numerical value is the highest numerical value for all DBP's in step (h) that bind to the target DNA sequence.
 3. The method of claim 1 wherein the DBP R_(b) numerical value determined in step (h) is at least 10,000.
 4. The method of claim 1 wherein the number of ZF domains, n_(d), is nine.
 5. The method of claim 1 wherein the rule s for assigning base-contacting amino acids at Z₁, Z₂ and Z₃ for each nucleotide block in step (e) are selected from rule set A.
 6. The method of claim 1 wherein the rules for assigning base-contacting amino acids at Z₁, Z₂ and Z₃ for each nucleotide block in step (e) are selected from rule set B.
 7. The method of claim 1 wherein rules for assigning base-contacting amino acids at Z₁, Z₂ and Z₃ for each nucleotide block in step (e) are a combination selected from rule sets A and B.
 8. A computer system for designing a DNA-binding protein (DBP), with multiple zinc-finger (ZF) domains connected by linker sequences, that binds selectively to a target DNA sequence within a given gene, each of said ZF domains having the formula A₁XCX₂₋₄CA₂A₃XFXZ₃XXZ₂LXZ₁HX₃₋₅H   (SEQ ID NO 3) and each of said linkers having the formula A₄A₅X₀₋₂EA₆ P   (SEQ ID NO 4), wherein (i) X is any amino acid; (ii) X₂₋₄ is a peptide from 2 to 4 amino acids in length; (iii) X₃₋₅ is a peptide from 3 to 5 amino acids in length; (iv) X₀₋₂ is a peptide from 0 to 2 amino acids in length; (iv) A₁ is selected from the group consisting of phenylalanine and tyrosine; (v) A₂ is selected from the group consisting of glycine and aspartic acid; (vi) A₃ is selected from the group consisting of lysine and arginine; (vii) A₄ is selected from the group consisting of threonine and serine; (viii) A₅ is selected from the group consisting of glycine and glutamic acid; (ix) A₆ is selected from the group consisting of lysine and arginine; (x) C is cysteine; (xi) F is phenylalanine; (xii) L is leucine; (xiii) H is histidine; (xiv) E is glutamic acid; (xv) P is proline; and (xvi) Z₁, Z₂ and Z₃ are the base-contacting amino acids, which computer system comprises a computer readable memory which executes a method for designing the DBP, wherein the method comprises the steps of: (a) setting a genome to be screened; (b) selecting the target DNA sequence in the genome for binding; (c) setting the number of ZF domains to n_(d); (d) dividing the target DNA sequence into nucleotide blocks wherein each block contains n_(z) nucleotides using a first routine where n_(z) is determined using the following relationship: n_(z)=3n_(d); (e) assigning base-contacting amino acids at Z₁, Z₂ and Z₃ to each ZF domain, according to the A Rules and/or B Rules set forth in Tables 1-3 of the specification, of a DBP which binds to the first nucleotide block from step (d) as numbered from the first 5′ nucleotide of the target gene sequence to generate a block-specific DBP and calculating the binding energy, Binding Energy_(block,) of each ZF domain of each such block-specific DBP as the product of the binding energies, Binding Energy_(domain,) of all ZF domains of the DBP, using the formula: Binding Energy_(domain)=(5×the number of hydrogen bonds)+(2×the number of H₂O contacts)+(the number of hydrophobic contacts); (f) subdividing the DBP from step (d) into blocks using a second routine to generate a subdivided DBP having three ZF domains; (g) screening the subdivided DBP from step (f) against the genome using a third routine to determine the number of binding sites in the genome for each subdivided DBP in the genome and assigning a binding energy for each such site using the following formula: Binding Energy_(site n)=(5×the number of hydrogen bonds)+(2×the number of H₂O contacts)+(the number of hydrophobic contacts); (h) calculating a ratio of binding energy, R_(b), using a fourth routine for each nucleotide block-specific DBP from step (e) using the following formula: R_(b)=Binding Energy_(block)/the sum of all Binding Energy_(site n)'s for all subdivided DBP's from step (g); (i) repeating steps (f) through (h) for each subdivided DBP wherein n_(d)≧4; (j) repeating steps (d) through (i) for each nucleotide block in the target DNA sequence containing n_(z) nucleotides; (k) rank-ordering R_(b) numerical values obtained from step (h); and (l) selecting a DBP with an acceptable R_(b) value.
 9. The computer system according to claim 8 wherein the DBP selected is that whose R_(b) numerical value is the highest numerical value for all DBP's in step (h) that bind to the target DNA sequence.
 10. The computer system according to claim 8 wherein the DBP R_(b) numerical value determined in step (h) is at least 10,000.
 11. The computer system according to claim 8 wherein the number of ZF domains, n_(d), is nine.
 12. The computer system according to claim 8 wherein the rules for assigning base-contacting amino acids at Z₁, Z₂ and Z₃ for each nucleotide block in step (e) are selected from rule set A.
 13. The computer system according to claim 8 wherein the rules for assigning base-contacting amino acids at Z₁, Z₂ and Z₃ for each nucleotide block in step (e) are selected from rule set B.
 14. The computer system according to claim 8 wherein the rules for assigning base-contacting amino acids at Z₁, Z₂ and Z₃ for each nucleotide block in step (e) are a combination selected from rule sets A and B. 